Method and system for detecting electrophysiological changes in pre-cancerous and cancerous tissue and epithelium

ABSTRACT

A method and system for determining a condition of a selected region of epithelial and stromal tissue, for example in the human breast. A plurality of electrodes are used to measure surface and transepithelial electropotential of breast tissue as well as surface electropotential and impedance at one or more locations and at several defined frequencies, particularly very low frequencies. An agent may be introduced into the region of tissue to enhance electrophysiological characteristics. Measurements made at ambient and varying suction and/or positive pressure applied to the epithelial tissue and/or positive pressure conditions applied to the breast are also used as a diagnostic tool. Tissue condition is determined based on the electropotential and impedance profile at different depths of the epithelium, stroma, tissue, or organ, together with an estimate of the functional changes in the epithelium due to altered ion transport and electrophysiological properties of the tissue. Devices for practicing the disclosed methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 60/673,448 filed Apr. 21, 2005, thedisclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the detection of abnormal orcancerous tissue, and more particularly, to the detection of changes inthe electrophysiological characteristics of abnormal or cancerous tissueand to changes in those electrophysiological characteristics related tothe functional, structural and topographic (the interaction of shape,position and function) relationships of the tissue during thedevelopment of malignancy. These measurements are made in the absenceand presence of pharmacological and hormonal agents to reveal andaccentuate the electrophysiological characteristics of abnormal orcancerous tissue.

Cancer is a leading cause of death in both men and women in the UnitedStates. Difficulty in detecting abnormal pre-cancerous or canceroustissue before treatment options become non-viable is one of the reasonsfor the high mortality rate. Detecting of the presence of abnormal orcancerous tissues is difficult, in part, because such tissues arelargely located deep within the body, thus requiring expensive, complex,invasive, and/or uncomfortable procedures. For this reason, the use ofdetection procedures is often restricted until a patient is experiencingsymptoms related to the abnormal tissue. Many forms of cancers ortumors, however, require extended periods of time to attain a detectablesize (and thus to produce significant symptoms or signs in the patient).It is often too late for effective treatment by the time the detectionis performed with currently available diagnostic modalities.

Breast cancer is the most common malignancy affecting women in theWestern World. The reduction in mortality for this common diseasedepends on early detection. The mainstay of early detection are X-raymammography and clinical breast examination. Both are fraught withproblems of inaccuracy. For example, mammography has a lower sensitivityin women with dense breasts, and is unable to discriminate betweenmorphologically similar benign or malignant breast lesions.

Clinical breast examinations are limited because lesions less than onecm are usually undetectable and larger lesions may be obscured bydiffuse nodularity, fibrocystic change, or may be too deep in the breastto enable clinical detection. Patients with positive mammographic orequivocal clinical findings often require biopsy to make a definitivediagnosis. Moreover, biopsies may be negative for malignancy in up to80% of patients.

Accordingly, mammography and clinical breast examination have relativelypoor specificity in diagnosing breast cancer. Therefore many positivemammographic findings or lesions detected on clinical breast examinationultimately prove to be false positives resulting in physical andemotional trauma for patients. Improved methods and technologies toidentify patients who need to undergo biopsy would reduce healthcarecosts and avoid unnecessary diagnostic biopsies.

Other technologies have been introduced in an attempt to improve on thediagnostic accuracy attainable with mammography and clinical breastexamination alone. Breast ultrasound is helpful in distinguishingbetween cystic or solid breast lesions and may be useful in guidingneedle or open biopsies. However, such techniques are unable todetermine whether a solid mass, or calcifications are benign ormalignant. Magnetic resonance imaging has been introduced in an attemptto improve on the accuracy of mammography. Its high cost and lowspecificity limit its general applicability for diagnosing and screeningfor breast cancer. Nuclear imaging with Positron Emission Tomogaphy(PET) has a lower sensitivity for small lesions, but is limited by cost.

It is also desirable to develop improved technology suitable fordiagnosing pre-cancerous tissue and cancer in other tissue types andelsewhere in the body, particularly methods and devices suitable forascertaining the condition of bodily ductal structures, e.g., theprostate, pancreas, etc., as well as the breast.

One proposed method for early detection of cancerous and pre-canceroustissue includes measuring of the electrical impedance of biologicaltissue. For example, U.S. Pat. No. 3,949,736 discloses a low-levelelectric current passed through tissue, with a measurement of thevoltage drop across the tissue providing an indirect indication of theoverall tissue impedance. This method teaches that a change in impedanceof the tissue is associated with an abnormal condition of the cellscomposing the tissue, indicating a tumor, carcinoma, or other abnormalbiological condition. This disclosure, however, does not discuss eitheran increase or decrease in impedance associated with abnormal cells, nordoes it specifically address tumor cells.

The disadvantage of this and similar systems is that the DC electricalproperties of the epithelium are not considered. Most commonmalignancies develop in an epithelium (the cell layer that lines ahollow organ, such as the bowel, or ductal structures such as the breastor prostate), that maintains a transepithelial electropotential. Earlyin the malignant process the epithelium loses its transepithelialpotential, particularly when compared to epithelium some distance awayfrom the developing malignancy. The combination of transepithelialelectropotential measurements with impedance are more accurate indiagnosing pre-cancerous and cancerous conditions.

Another disadvantage of the above referenced system is that thefrequency range is not defined. Certain information is obtained aboutcells according to the range of frequencies selected. Differentfrequency bands may be associated with different structural orfunctional aspects of the tissue. See, for example, F. A. Duck, PhysicalProperties of Tissues, London: Academic Press, 2001; K. R. Foster, H. P.Schwan, Dielectric properties of tissues and biological materials: acritical review, Crit. Rev. Biomed. Eng., 1989, 17(1): 25-104. Forexample at high frequencies such as greater than about 1 GHz molecularstructure has a dominating effect on the relaxation characteristics ofthe impedance profile. Relaxation characteristics include the delay inthe response of a tissue to a change in the applied electric field. Forexample, an applied AC current results in voltage change across thetissue which will be delayed or phase shifted, because of the impedancecharacteristics of the tissue. Relaxation and dispersion characteristicsof the tissue vary according to the frequency of the applied signal.

At lower frequencies, such as less than about 100 Hz, or the so calledα-dispersion range, alterations in ion transport and chargeaccumulations at large cell membrane interfaces dominate the relaxationcharacteristics of the impedance profile. In the frequency range betweena few kHz and about 1 MHz, or the so-called β-dispersion range, cellstructure dominates the relaxation characteristics of the epithelialimpedance profile. Within this range at low kHz frequencies, most of theapplied current passes between the cells through the paracellularpathway and tight junctions. At higher, frequencies in the β-dispersionrange the current can penetrate the cell membrane and therefore passesboth between and through the cells, and the current density will dependon the composition and volume of the cytoplasm and cell nucleus.Characteristic alterations occur in the ion transport of an epitheliumduring the process of malignant transformation affecting the impedancecharacteristics of the epithelium measured at frequencies in theα-dispersion range. Later in the malignant process, structuralalterations with opening of the tight junctions and decreasingresistance of the paracellular pathways, together with changes in thecomposition and volume of the cell cytoplasm and nucleus, affect theimpedance measured in the β-dispersion range.

Another disadvantage with the above referenced system is that thetopography of altered impedance is not examined. By spacing themeasuring electrodes differently the epithelium can be probed todifferent depths. The depth that is measured by two surface electrodesis approximately half the distance between the electrodes. Thereforeelectrodes 1 mm apart will measure the impedance of the underlyingepithelium to a depth of approximately 500 microns. It is known, forexample, that the thickness of bowel epithelium increases at the edge ofa developing tumor to 1356±208μ compared with 716±112μ in normal bowel.D. Kristt, et al. Patterns of proliferative changes in crypts borderingcolonic tumors: zonal histology and cell cycle marker expression.Pathol. Oncol. Res 1999; 5(4): 297-303. Thickening of the ductalepithelium of the breast is also observed as ductal carcinoma in-situdevelops. By comparing the measured impedance between electrodes spacedapproximately 2.8 mm apart and compared with the impedance of electrodesspaced approximately 1.4 mm apart, information about the deeper andthickened epithelium may be obtained. See, for example, L. Emtestam & S.Ollmar. Electrical impedance index in human skin: measurements afterocclusion, in 5 anatomical regions and in mild irritant contactdermatitis. Contact Dermatitis 1993; 28(2): 104-108.

Another disadvantage of the above referenced methods is that they do notprobe the specific conductive pathways that are altered during themalignant process. For example, potassium conductance is reduced in thesurface epithelium of the colon early in the malignant process. By usingelectrodes spaced less than 1 mm apart with varying concentrations ofpotassium chloride the potassium conductance and permeability may beestimated in the surface epithelium at a depth from less than 500μ tothe surface.

A number of non-invasive impedance imaging techniques have beendeveloped in an attempt to diagnose breast cancer. Electrical impedancetomography (EIT) is an impedance imaging technique that employs a largenumber of electrodes placed on the body surface. The impedancemeasurements obtained at each electrode are then processed by a computerto generate a 2 dimensional or 3 dimensional reconstructed tomographicimage of the impedance and its distribution in 2 or 3 dimensions. Thisapproach relies on the differences in conductivity and impedivitybetween different tissue types and relies on data acquisition and imagereconstruction algorithms which are difficult to apply clinically.

The majority of EIT systems employ “current-driving mode,” which appliesa constant AC current between two or more current-passing electrodes,and measures the voltage drop between other voltage-sensing electrodeson the body surface. Another approach is to use a “voltage-drivingapproach,” which applies a constant AC voltage between two or morecurrent-passing electrodes, and then measures the current at othercurrent-sensing electrodes. Different systems vary in the electrodeconfiguration, current or voltage excitation mode, the excitation signalpattern, and AC frequency range employed.

Another disadvantage with using EIT to diagnose breast cancer is theinhomogeneity of breast tissue. The image reconstruction assumes thatcurrent passes homogeneously through the breast tissue which is unlikelygiven the varying electrical properties of different types of tissuecomprising the breast. In addition image reconstruction depends upon thecalculation of the voltage distribution on the surface of the breastfrom a known impedance distribution (the so called forward problem), andthen estimating the impedance distribution within the breast from themeasured voltage distribution measured with surface electrodes (theinverse problem). Reconstruction algorithms are frequently based onfinite element modeling using Poisson's equation and with assumptionswith regard to quasistatic conditions, because of the low frequenciesused in most EIT systems.

Other patents, such as U.S. Pat. Nos. 4,955,383 and 5,099,844, disclosethat surface electropotential measurements may be used to diagnosecancer. Empirical measurements, however, are difficult to interpret anduse in diagnosis. For example, the above referenced inventions diagnosecancer by measuring voltage differences (differentials) between oneregion of the breast and another and then comparing them withmeasurements in the opposite breast. Changes in the measured surfacepotential may be related to differences in the impedance characteristicsof the overlying skin. This fact is ignored by the above referenced andsimilar inventions, resulting in a diagnostic accuracy of 72% or less.J. Cuzick et al. Electropotential measurements as a new diagnosticmodality for breast cancer. Lancet 1998; 352(9125): 359-363; M. Faupelet al. Electropotential evaluation as a new technique for diagnosingbreast lesions. Eur. J. Radiol. 1997; 24 (1): 33-38. Neither ACimpedance, or surface DC measurement approaches, measure thetransepithelial breast DC potential or AC impedance characteristics ofthe breast epithelium.

Other inventions that use AC measurement, such as U.S. Pat. No.6,308,097, also have a lower accuracy than may be possible with acombination of DC potential measurements and AC impedance measurements,that also measure the transepithelial electrical properties of mammaryepithelium. Electrical impedance scanning (EIS) also known as electricalimpedance mapping (EIM) avoids the limitations of complex imagereconstruction encountered with EIT. The above referenced systemdiagnoses cancer by only measuring decreased impedance (increasedconductance) and changes in capacitance over a cancer. It does notmeasure the mammary transepithelial impedance characteristics of thebreast. There are several other limitations to this approach.Inaccuracies may occur because of air bubbles. Underlying bones, costalcartilages, muscle and skin may result in high conductance regions,which produce false positives. Depth of measurement is limited to 3-3.5cm, which will result in false negatives for lesions on the chest wall.It is also not possible to localize lesions using this approach.

Another potential source of information for the detection of abnormaltissue is the measurement of transport alterations in the epithelium.Epithelial cells line the surfaces of the body and act as a barrier toisolate the body from the outside world. Not only do epithelial cellsserve to insulate the body, but they also modify the body's environmentby transporting salts, nutrients, and water across the cell barrierwhile maintaining their own cytoplasmic environment within fairly narrowlimits. One mechanism by which the epithelial layer withstands theconstant battering is by continuous proliferation and replacement of thebarrier. This continued cell proliferation may partly explain why morethan 80% of cancers are of epithelial cell origin. Moreover, given theirspecial abilities to vectorially transport solutes from blood to outsideand vice versa, it appears that a disease process involving alteredgrowth regulation may have associated changes in transport properties ofepithelia.

It is known that the addition of serum to quiescent fibroblasts resultsin rapid cell membrane depolarization. Cell membrane depolarization isan early event associated with cell division. Depolarization induced bygrowth factors appears biphasic in some instances but cell division maybe stimulated without depolarization. Cell membrane depolarization istemporally associated with Na⁺ influx, and the influx persists afterrepolarization has occurred. Although the initial Na⁺ influx may resultin depolarization, the increase in sodium transport does not cease oncethe cell membrane has been repolarized, possibly due to Na/K ATPase pumpactivation. Other studies also support the notion that Na⁺ transport isaltered during cell activation. In addition to altered Na⁺-transport,K⁺-, and Cl⁻-transport is altered during cell proliferation.

A number of studies have demonstrated that proliferating cells arerelatively depolarized when compared to those that are quiescent ornon-dividing. Differentiation is associated with the expression ofspecific ion channels. Additional studies indicate that cell membranedepolarization occurs because of alterations in ionic fluxes,intracellular ionic composition and transport mechanisms that areassociated with cell proliferation.

Intracellular Ca²⁺ (Ca²⁺ _(i)) and pH (pH_(i)) are increased by mitogenactivation. Cell proliferation may be initiated following the activationof phosphatidylinositol which releases two second messengers,1,2-diacylglycerol and inosotol-1,4,5-triphosphate, which triggers Ca²⁺_(i) release from internal stores. Ca²⁺ _(i) and pH_(i) may then alterthe gating of various ion channels in the cell membrane, which areresponsible for maintaining the voltage of the cell membrane. Therefore,there is the potential for interaction between other intracellularmessengers, ion transport mechanisms, and cell membrane potential. Moststudies have been performed in transformed and cultured cells and not inintact epithelia during the development of cancer.

It was known for some time that cancer cells are relatively depolarizedcompared with non-transformed cells. It has been suggested thatsustained cell membrane depolarization results in continuous cellularproliferation, and that malignant transformation results as aconsequence of sustained depolarization and a failure of the cell torepolarize after cell division. C. D. Cone Jr., Unified theory on thebasic mechanism of normal mitotic control and oncogenesis. J. Theor.Biol. 1971; 30(1): 151-181; C. D. Cone Jr., C. M. Cone. Induction ofmitosis in mature neurons in central nervous system by sustaineddepolarization. Science 1976; 192(4235): 155-158; C. D. Cone, Jr. Therole of the surface electrical transmembrane potential in normal andmalignant mitogenesis. Ann. N.Y. Acad. Sci. 1974; 238: 420-435. A numberof studies have demonstrated that cell membrane depolarization occursduring transformation and carcinogenesis. Other studies havedemonstrated that a single ras-mutation may result in altered iontransport and cell membrane depolarization. Y. Huang, S. G. Rane, Singlechannel study of a Ca(2+)-activated K+ current associated with rasinduced cell transformation. J. Physiol. 1993; 461: 601-618. Forexample, there is a progressive depolarization of the colonocyte cellmembrane during 1,2 dimethylhydrazine (DMH)-induced colon cancer in CF₁mice. The V_(A) (apical membrane voltage) measured with intracellularmicroelectrodes in histologically “normal” colonic epitheliumdepolarized from −74.9 mV to −61.4 mV after 6 weeks of DMH treatment andto −34 mV by 20 weeks of treatment. The cell membrane potential in abenign human breast epithelial cell line (MCF-10A) was observed to be−50±4 mV (mean±SEM) and was significantly depolarized at −35±1 mV(p<0.002) in the same cell line after ras-transformation (the MCF-10ATcell line).

While epithelial cells normally maintain their intracellular sodiumconcentration within a narrow range, electronmicroprobe analysissuggests that cancer cells exhibit cytoplasmic sodium/potassium ratiosthat are three to five times greater than those found in theirnon-transformed counterparts. These observations partly explain theelectrical depolarization observed in malignant or pre-malignanttissues, because of the loss of K⁺ or Na⁺ gradients across the cellmembrane.

In addition to cell membrane depolarization, and altered intracellularionic activity, other studies have shown that there may be a decrease inelectrogenic sodium transport and activation of non-electrogenictransporters during the development of epithelial malignancies. Thesechanges may affect or occur as a consequence of altered intracellularionic composition.

In addition to cell membrane depolarization, and altered intracellularionic activity, other studies have shown that there may be a decrease inelectrogenic sodium transport and activation of non-electrogenictransporters during the development of epithelial malignancies. Thesechanges may occur as a consequence of altered intracellular ioniccomposition. Other specific ion transport alterations have beendescribed in colon, prostate, breast, uterine cervix, melanoma,urothelium, and pancreas during proliferation, differentiation,apoptosis, and carcinogenesis.

Apoptosis or physiological cell death is down-regulated during thedevelopment of malignancy. Ion transport mechanisms affected byapoptosis include the influx of Ca²⁺ non-selective Ca²⁺-permeable cationchannels, calcium-activated chloride channels, and K⁺—Cl⁻ cotransport.J. A. Kim et al. Involvement of Ca2+ influx in the mechanism oftamoxifen-induced apoptosis in Hep2G human hepatoblastoma cells. CancerLett. 1999; 147(1-2): 115-123; A. A. Gutierrez et al. Activation of aCa2+-permeable cation channel by two different inducers of apoptosis ina human prostatic cancer cell line. J. Physiol. 1999; 517 (Pt. 1):95-107; J. V. Tapia-Vieyra, J. Mas-Oliva. Apoptosis and cell deathchannels in prostate cancer. Arch. Med. Res. 2001; 32(3): 175-185; R. C.Elble, B. U. Pauli. Tumor Suprression by a ProapoptoticCalcium-Activated Chloride Channel in Mammary Epithelium. J. Biol. Chem.2001; 276(44): 40510-40517.

Loss of cell-to-cell communication occurs during carcinogenesis. Thisresults in defective electrical coupling between cells, which ismediated via ions and small molecules through gap junctions, which inturn influences the electrical properties of epithelia.

Epithelial cells are bound together by tight junctions, which consist ofcell-to-cell adhesion molecules. These adhesion proteins regulate theparacellular transport of molecules and ions between cells and aredynamic structures that can tighten the epithelium, preventing themovement of substances, or loosen allowing substances to pass betweencells. Tight junctions consist of integral membrane proteins, claudins,occludins and JAMs (junctional adhesion molecules). Tight junctions willopen and close in response to intra and extracellular stimuli.

A number of substances will open or close tight junctions. Theproinflammatory agent TGF-alpha, cytokines, IGF and VEGF opens tightjunctions. Zonula occludens toxin, nitric oxide donors, and phorbolesters also reversibly open tight junctions. Other substances closetight junctions including calcium, H2 antagonists and retinoids. Varioushormones such as prolactin and glucocorticoids will also regulate thetight junctions. Other substances added to drug formulations act asnon-specific tight junction modulators including chitosan and wheat germagglutinin.

The above referenced substances and others may act directly orindirectly on the tight junction proteins, which are altered duringcarcinogenesis. For example claudin-7 is lost in breast ductalepithelium during the development of breast cancer. The response of thetight junctions varies according to the malignant state of theepithelium and their constituent proteins. As a result the opening orclosing of tight junctions is affected by the malignant state of theepithelium.

Polyps or overtly malignant lesions may develop in a background ofdisordered proliferation and altered transepithelial ion transport.Experimental animal studies of large bowel cancer have demonstrated thattransepithelial depolarization is an early feature of the pre-malignantstate. In nasal polyp studies, the lesions had a higher transepithelialpotential, but these lesions were not pre-malignant in the same sense asan adenomatous or pre-malignant colonic polyp, that are usuallydepolarized. Electrical depolarization has been found in biopsies ofmalignant breast tissue. Recently alterations in impedance have beenfound to be associated with the pre-malignant or cancerous state inbreast and bowel.

It has been discovered that transepithelial depolarization was aspecific event associated with colonic carcinogenesis in CF₁ mice. Themore susceptible site, the distal colon, underwent about a 30% decreasein transepithelial potential (V_(T)) after only four weeks of carcinogentreatment. This was before histological changes developed. Anon-specific cytotoxic agent (5-fluorouracil), administered over thesame period did not cause a reduction in V_(T) in the same model. Thereduction in V_(T) was confirmed in a subsequent study where almost a60% reduction was observed after carcinogen treatment. It has also beendiscovered that, although V_(T) is invariably higher when measured invivo, the “premalignant” colonic epithelium is usually depolarized whencompared to normal colon.

DC electrical potential alterations have been used to diagnosenon-malignant conditions such as cystic fibrosis, cancer in animalmodels, human cells or tissue and in man. Differences in impedancebetween normal tissue and cancer have been described in animal models invitro human tissue in vitro and have been applied to in vivo cancerdiagnosis.

DC potential measurements have not been combined with impedancemeasurements to diagnose cancer because the electrophysiologicalalterations that accompany the development of cancer have not been wellunderstood or fully characterized. Surface measurements of potential orimpedance are not the same as measurements performed across the breastepithelium, and described below, where electrical contact is madebetween the luminal surface of the duct and the overlying skin.Transepithelial depolarization is an early event during carcinogenesis,which may affect a significant region of the epithelium (a “fielddefect”). This depolarization is accompanied by functional changes inthe epithelium including ion transport and impedance alterations. Earlyon in the process these take the form of increased impedance because ofdecreased specific electrogenic ion transport processes. As the tumorbegins to develop in the pre-malignant epithelium, structural changesoccur in the transformed cells such as a breakdown in tight junctionsand nuclear atypia. The structural changes result in a marked reductionin the impedance of the tumor. The pattern and gradient of electricalchanges in the epithelium permit the diagnosis of cancer from acombination of DC electrical and impedance measurements.

Another reason that DC electropotential and impedance measurements havenot been successfully applied to cancer diagnosis is thattransepithelial potential and impedance may be quite variable and areaffected by the hydration state, dietary salt intake, diurnal orcyclical variation in hormonal level or non-specific inflammatorychanges and other factors. In the absence of knowledge about thephysiological variables which influence transepithelial potential andimpedance these kind of measurement may not be completely reliable todiagnose pre-malignancy or cancer.

Furthermore, a detailed understanding of the functional andmorphological alterations that occur during carcinogenesis permitsappropriate electrical probing for a specifically identified iontransport change that is altered during cancer development. For exampleknowledge that electrogenic sodium absorption is altered during cancerdevelopment in breast epithelium permits the use of sodium channelblockers (amiloride) or varying sodium concentration in the ECM(electroconductive medium) to examine whether there is an inhibitablecomponent of sodium conductance. By varying the depth of the measurement(by measuring the voltage drop across differently space electrodes), itis possible to obtain topographic and depth information about thecancerous changes in the epithelium. Using a combination of low and highfrequency sine waves probing at different depths we are able tocorrelate the functional and morphological (structural) changes atdifferent depths, with the impedance profile of the tissue.

The diagnostic accuracy of current technology using DC electropotentialsor impedance alone have significant limitations. Sensitivity andspecificity for DC electrical measurements in the breast have beenreported as 90% and 55% respectively and 93% and 65% for impedancemeasurements. This would result in an overall diagnostic accuracy ofbetween 72-79%, which is probably too low to result in widespreadadoption. The measurement of ductal transepithelial DC potential, ductaltransepithelial AC impedance spectroscopy alone, or the combination ofDC electrical potentials and impedance spectroscopy will result in adiagnostic accuracy of greater than 90%, which will lead to improvedclinical utility.

Breast cancer is thought to originate from epithelial cells in theterminal ductal lobular units (TDLUs) of mammary tissue. These cellsproliferate and have a functional role in the absorption and secretionof various substances when quiescent and may produce milk whenlactating. Functional alterations in breast epithelium have largely beenignored as a possible approach to breast cancer diagnosis. Breastepithelium is responsible for milk formation during lactation. Everymonth pre-menopausal breast epithelium undergoes a “rehearsal” forpregnancy with involution following menstruation. The flattenedepithelium becomes more columnar as the epithelium enters the lutealphase from the follicular phase. In addition, duct branching and thenumber of acini reach a maximum during the latter half of the lutealphase. Just before menstruation apoptosis of the epithelium occurs andthe process starts over again unless the woman becomes pregnant.

Early pregnancy and lactation may be protective against breast cancerbecause they result in a more differentiated breast epithelium which isless susceptible to carcinogenic influences whether estrogen or otherenvironmental factors. It therefore seems that differentiated breastepithelium is less likely to undergo malignant change. Differentiatedepithelium has a distinct apical and basolateral membrane domain toenable it to maintain vectorial transport function (the production ofmilk). In addition, differentiated cells maintain a higher cell membranepotential to transport various ions, lactulose and other substances inand out of the duct lumen. In contrast, more proliferative epithelialcells have depolarized cell membranes and are less able to maintainvectorial ion transport. Recently the epithelial Na⁺ channel (ENaC) andthe cystic fibrosis transmembrane conductance regulator (CFTR) have beenidentified in mammary epithelium and both localized on the apical, orluminal side, of the epithelium. These two transporters can be probedfor by using amiloride, a blocker of the ENaC, or by opening up Cl⁻channels regulated by CFTR using-cAMP.

For example, 20 μM luminal amiloride depolarized the transepithelialpotential from −5.9±0.5 mV (mean±SEM) by +3.1±0.5 mV. Forskolin (10M),which raises cAMP and opens Cl⁻ channels via the CFTR hyperpolarized thebreast epithelium by −2.2±0.1 mV. These changes were accompanied by anincrease (17%) and subsequent decrease (19%) in transepithelialresistance respectively. In transformed breast epithelium the ENaC isdown-regulated, whereas Cl⁻ secretion may increase, similar toobservations reported for carcinoma of the cervix. Non-lactating breastepithelium has relatively leaky tight junctions. This results in aparacellular shunt current, which hyperpolarizes the apical membrane ofthe epithelial cell. The larger the shunt current the morehyperpolarized the apical membrane and therefore the epitheliumdepolarizes since:TEP=V _(BL) −V _(A) and i=TEP/R _(S); where

TEP=Transepithelial potential;

V_(BL)=voltage of the basolateral membrane;

V_(A)=voltage of the apical membrane;

i=shunt current; and

R_(S)=paracellular (shunt) resistance.

Evidence that breast carcinogenesis may be associated with functionalincompetence of breast epithelium also comes from a number of othersources. Some transgenic strains of mice have defective lactation. Thetransgenic src mouse which develops hyperplastic alveolar nodules,otherwise develops a normal mammary tree but has defective lactation.The notch4 and TGFβ transgenic mouse also demonstrate defectivelactation. Cyclin D1 females have persistent lactation 6-9 months afterweaning, and TGFα mice, which have a defect in apoptosis and fail toundergo epithelial regression develop hypersecretion. These data suggestthat there is a relationship between epithelial function and geneticexpression which affects proliferation and tumor development.

Breast cysts occur in 7% of the female population and are thought todevelop in the TDLUs. Apocrine cysts have a higher potassium contentthan simple cysts. Apocrine cysts may be associated with the subsequentdevelopment of breast cancer. There may therefore be a fundamentalchange in the epithelium at risk for breast cancer development with aredistribution of electrolyte content across the cell membrane resultingin altered cyst electrolyte content and cell membrane depolarization.Although it is commonly known that during lactation the breasttransports lactulose, proteins, fatty acids, immunoglobulinscholesterol, hormones, ions and water across the ductal and lobularepithelium and actively secretes milk, it is less widely appreciatedthat in the non-pregnant and non-lactating state the breast, throughoutlife exhibits excretory and absorptive function. The difference betweenthe lactating and the non-lactating breast being of degree and thechemical constitution of the nipple duct fluid. Ductal secretions havebeen analyzed to diagnose biological conditions of the breast.

A number of approaches have been used to obtain ductal fluid, includinga suction cup to obtained pooled secretions; nipple aspiration fluid(NAF), and more recently, cannulation of one of the 6-12 ducts that openonto the nipple surface. Substances and cells within the duct fluid maytherefore be accessed to identify abnormalities that may be associatedwith the diseased state of the breast. One disadvantage of the abovereferenced approaches is the difficulty in obtaining adequate NAF orlavage fluid to perform analysis. Another disadvantage has been theinability to identify or cannulate the ducts where an abnormality in thefluid or cells may be identified.

Hung (U.S. Pat. No. 6,314,315) has suggested an electrical approach toidentify ductal orifices on the nipple surface. In that disclosure it istaught that DC potential or impedance measurement may facilitate theidentification of openings or orifices on the surface of the nipple.However, it is not taught that the characteristics of the DC electricalsignal or impedance may characterize the condition of the breast.Moreover, it is not taught that breast transepithelial DC measurements,transepithelial AC impedance spectroscopy, alone or in combination maybe used to diagnose breast cancer.

Ionic gradients exist between the fluid secretions within the breastducts and the plasma. For example, it is known that the nipple aspiratefluid has a sodium concentration [Na⁺] of 123.6±33.8 mEq/l(mean±standard deviation) compared with a serum [Na⁺] of approximately150 mEq/l (Petrakis1). Nulliparous women have NAF [Na⁺] that areapproximately 10 mEq/l higher than parous women, but still significantlybelow serum levels. Similarly potassium concentration [K⁺] issignificantly higher at 13.5±7.7 mEq/l in parous women and 12.9±6.0mEq/l in nulliparous women compared with serum levels of [K⁺] ofapproximately 5.0 mEq/l. Other investigators have reported lower NAF[Na⁺] of 53.2 mEq/l suggesting that significant ionic gradients can beestablished between the plasma and duct lumen in non-lactating breast.In pregnancy these gradients are even higher for sodium with a [Na⁺] of8.5±0.9 mEq/l reported in milk which is almost 20 fold lower thanplasma. Chloride concentration [Cl⁻] in milk is almost one tenth of theconcentration found in plasma with values of 11.9±0.5 mM reported.Although [Na⁺] and [Cl⁻] levels in ductal secretions rise and the [K⁺]falls following the cessation of lactation, significant ionic gradientsare maintained between the duct lumen and plasma.

Furthermore, in women undergoing ovulatory cycles during lactationdistinct changes have been observed in the ion and lactuloseconcentrations of breast milk. The first change occurs 5-6 days beforeovulation and the second 6-7 days after ovulation. During these periods[Na⁺] and [Cl⁻] increased more than two-fold and [K⁺] decreasedapproximately 1.5-fold. It is unclear whether changes in estrogen orprogesterone levels before and after ovulation are affecting the ioncomposition of milk. However, it is known that alterations in the ioniccomposition of milk influences the transepithelial electrical potentialas measured in mammals.

Furthermore, it is known that various hormones affect breast epithelialion transport. For example, prolactin decreases the permeability of thetight-junctions between breast epithelial cells, stimulates mucosal toserosal Na⁺ flux, upregulates Na⁺:K⁺:2Cl⁻ cotransport and increases the[K⁺] and decreases the [Na⁺] in milk. Glucocorticoids control theformation of tight-junctions increasing transepithelial resistance anddecreasing epithelial permeability. Administration of cortisol intobreast ducts late in pregnancy has been shown to increase the [K⁺] anddecrease [Na⁺] of ductal secretions. Progesterone inhibitstight-junction closure during pregnancy and may be responsible for thefluctuations in ductal fluid electrolytes observed during menstrualcycle in non-pregnant women, and discussed above. Estrogen has beenobserved to increase cell membrane and transepithelial potential and maystimulate the opening of K⁺-channels in breast epithelial cells. Thehormones mentioned above vary diurnally and during menstrual cycle. Itis likely that these variations influence the functional properties ofbreast epithelium altering the ionic concentrations within the lumen,the transepithelial potential and impedance properties, which aredependent upon the ion transport properties of epithelial cells and thetranscellular and paracellular conductance pathways.

Accordingly, these variations can be used as diagnostic indicia ofchanges to breast tissue, which have to date yet to be exploited. Thus,there remains a need for effective and practical methods for detectingabnormal breast tissue as well as other epithelial and/or ductal tissue.

The disclosures of the following patent applications, each to Richard J.Davies, the inventor herein, are hereby incorporated by referenceherein: U.S. patent application Ser. No. 10/151,233, filed May 20, 2002,entitled “Method and System for Detecting Electrophysiological Changesin Pre-Cancerous and Cancerous Tissue”; U.S. patent application Ser. No.10/717,074, filed Nov. 19, 2003, entitled “Method And System ForDetecting Electrophysiological Changes In Pre-Cancerous And CancerousBreast Tissue And Epithelium”; and U.S. patent application Ser. No.10/716,789, filed Nov. 19, 2003, entitled “ElectrophysiologicalApproaches To Assess Resection and Tumor Ablation Margins and ResponsesTo Drug Therapy”.

SUMMARY OF THE INVENTION

One aspect of the invention provides a method for determining acondition of a region of epithelial tissue, for example epithelialbreast tissue comprising: establishing a connection between a firstelectrode and the epithelial tissue of the nipple of a breast; placing asecond electrode in contact with the surface of the breast; establishinga signal between the first and second electrodes; establishing that thenipple ducts of the breast are open; measuring between the first andsecond electrode (1) a DC potential; and (2) impedance at about 5different frequencies in the range of about 10 Hz to about 200 Hz andimpedance at from about 5 to about 50 different frequencies in the rangeof about 0.1 Hz to about 10 Hz; and determining the condition of theregion of epithelial tissue based on the DC potential and impedancemeasurements between the first and second electrode.

In another aspect of the invention there is provided a method fordetermining a condition of a region of epithelial tissue, for example,epithelial breast tissue comprising: placing over the nipple of a breasta cup having an interior, and first and second openings, and anelectrode disposed within the interior, the cup having a source ofsuction in communication with the first opening, the second openinghaving been placed over the nipple; establishing an electricalconnection between the electrode and the epithelial lining of themammary ducts, which are in continuity with the surface of the nipple ofa breast through the duct openings (orifices); placing a secondelectrode in electrical contact with the surface of the breast;establishing a signal between the first and second electrodes;establishing that the nipple ducts of the breast are open; measuring aDC potential between the first and second electrode; applying suction tothe cup sufficient to effect ductal collapse in a normal ornon-malignant duct; and measuring impedance at about 5 differentfrequencies in the range of about. 10 Hz to about 200 Hz and impedanceat from about 5 to about 50 different frequencies in the range of about0.1 Hz to about 10 Hz; and altering the suction level and againmeasuring impedance at about 5 different frequencies of the range ofabout 10 Hz to about 200 Hz and impedance at from about 5 to about 50different frequencies in the range of about 0.1 Hz to about 10 Hz; anddetermining the condition of the region of epithelial tissue within theducts and lobules of the breast, based on the DC potential, and theimpedance measurements under varying pressure conditions.

In yet another embodiment of the invention there is provided A methodfor determining a condition of a region of epithelial or stromal tissuecomprising: A. measurement of an impedance spectrum over a region oftissue where an abnormality is suspected (suspicious region); B.measurement of an impedance spectrum over a region of tissue where noabnormality is suspected (control region); C. application of mechanicalpressure or compression over the suspicious region; D. measurement ofthe impedance spectrum in the suspicious region following theapplication of mechanical pressure; E. application of mechanicalpressure or compression over the control region; F. comparison of theimpedance profile before and after compression in the suspicious region;G. comparison of the impedance profile before and after compression inthe control region; and H. comparison of the impedance profile of thesuspicious region to the control region.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one embodiment of the inventionand together with the description, serve to explain the principles ofthe invention.

FIG. 1 is a schematic diagram of a DC and AC impedance measuring device,consistent with an embodiment of the present invention;

FIG. 2 illustrates an exemplary embodiment of a device suitable for usewith systems and methods consistent with the present invention;

FIG. 3 illustrates an exemplary embodiment of a surface measurementprobe suitable for use with systems and methods consistent with thepresent invention;

FIG. 4 illustrates an exemplary embodiment of a nipple electrodesuitable for use with systems and methods consistent with the presentinvention;

FIG. 5 illustrates an exemplary embodiment of a ductal electrode probesuitable for use with systems and methods consistent with the presentinvention;

FIG. 6 illustrates varying ionic content and the effect ontransepithelial conductance in human breast epithelium;

FIG. 7 illustrates measurements of cell membrane potential in humanbreast epithelial cells;

FIG. 8 illustrates the effect of increasing estradiol concentrations onthe transepithelial potential in benign and malignant breast epithelia;

FIG. 9 illustrates conductance and the electropotential measurementsmade over the surface of the breast in women with and without breastcancer;

FIG. 10 illustrates the measurement of electropotentials at the surfaceof the breast, and variation of the measurement during menstrual cycle;

FIG. 11 illustrates electrophysiological changes that occur within theductal epithelium during the development of breast cancer;

FIG. 12 illustrates changes in the short circuit current of humanepithelium exposed to a potassium channel blocker (TEA) or varyingconcentrations of potassium;

FIG. 13 illustrates how the information obtained in FIG. 12 may be usedto plot the potassium gradient against the change in short circuitcurrent.

FIG. 14 illustrates multiple Nyquist impedance plots from human breastsaccording to the present invention.

FIG. 15 illustrates the impedance profile for a patient with ahemorrhagic cyst.

FIG. 16 illustrates a Bode plot of impedance data comparing patientswith fibrocystic disease (0465) and breast cancer (0099).

FIG. 17 illustrates the same data as in FIG. 16 plotted as a Nyquistplot.

FIG. 18 illustrates the impedance spectra data curve for breast cancertissue added to the curves of FIG. 17.

FIG. 19 illustrates the effects of altering the level of suction appliedto a nipple cup electrode on a normal breast.

FIG. 20 illustrates the effects of altering the level of suction appliedto a nipple cup electrode on a breast in which malignancy is present.

FIG. 21 illustrates the method for estimating impedance for the highsuction curve associated with cancer in FIG. 20.

FIG. 22 illustrates the impedance profiles of a fibroadenoma andcarcinoma.

FIG. 23 illustrates the impedance profile of a normal duct followingcompression.

FIG. 24 illustrates the same results as in FIG. 23 with an expandedrange for the X-axis.

FIG. 25 illustrates the impedance profile of a normal duct followingcompression and release.

FIG. 26 illustrates the impedance profile of a breast cyst followingcompression.

FIG. 27 illustrates the impedance profile following compression offibrocystic breast tissue.

FIG. 28 illustrates the impedance profile following compression offibroadenoma in breast tissue.

FIG. 29 illustrates the impedance profile following compression of amore typical fibroadenoma in breast tissue.

DETAILED DESCRIPTION

Reference will now be made in detail to an embodiment of the invention,an example of which is illustrated in the accompanying drawings.Wherever possible, the same reference numbers will be used throughoutthe drawings to refer to the same or like parts.

The present invention overcomes problems and inadequacies associatedwith prior methods used for characterizing abnormal or cancerousepithelial tissue. In summary, various embodiments of the presentinvention use DC and/or impedance measurements, under ambient and/orvariable suction, that pass the current or signal across the breastepithelium and tumor using specially constructed electrodes. For examplea nipple electrode may be used to measure the voltage and/or impedancebetween ductal epithelium, surrounding breast tissue, skin and surfaceor other electrode. The nipple electrode may also be used to pass thecurrent along the ductal system of the breast. Another type of electrodemay be used to measure the voltage and/or impedance signal, and/or passa current and measure the signal at the individual ductal orifices atthe nipple surface. Another type of electrode may be used to measure thevoltage and/or impedance signal, and/or pass a current and measure thesignal within individual ducts using a modified ductal probe orductoscope which may have one or more electrodes attached to it. All ofthese electrodes may be used individually, in combination with oneanother, or with a surface probe or electrodes. Additionally DC andimpedance measurements will be used in combination to more adequatelycharacterize abnormal or cancerous tissues. DC measurements provideinformation about the functional state of the epithelium and can detectearly pre-malignant changes and an adjacent malignancy. In particular,impedance measurements at several frequencies in specifically definedranges using differently spaced electrodes provide depth and topographicinformation to give both structural (high frequency range) andfunctional (low frequency range) information about the tissue beingprobed. Abnormal or cancerous tissue can be detected and characterizedby detecting and measuring transport alterations in epithelial tissues,using ionic substitutions and/or pharmacological and hormonalmanipulations to determine the presence of abnormal pre-cancerous orcancerous cells. A baseline level of transepithelial DC potential,impedance or other electrophysiological property that is sensitive toalterations in transport in epithelia is measured in the tissue to beevaluated. An agent may be introduced to enhance the transport or makeit possible to detect the transport alteration. The transepithelial DCpotential and/or impedance of the tissue (or other electrophysiologicalproperty that may reflect or make it possible to detect alterations inthe transport) are then measured. Based on the agent introduced and themeasured electrophysiological parameter, the condition of the tissue isdetermined.

A method and system are provided for determining a condition of aselected region of breast epithelial tissue. At least twocurrent-passing electrodes are located in contact with a first surfaceof the selected region of the tissue. Alternatively the current passingelectrodes may pass current across the tissue or epithelium as forexample between the nipple ducts, ductal lumen, epithelium, breastparenchyma and surface of the breast. Alternatively, the ducts may beaccessed by a central duct catheter or ductoscope. A plurality ofmeasuring electrodes are located in contact with the first surface ofthe breast as well. Initially, one or more of the measuring electrodesis used to measure the DC potential referenced to another electrode, orreference point. A signal is established between the current-passingelectrodes. Impedance, associated with the established signal, ismeasured by one or more of the measuring electrodes. Alternatively athree-electrode system may be used for measurements whereby oneelectrode is used for both current injection and voltage recording. Anagent is introduced into the region of tissue. The condition of thetissue is determined based on the effect of the agent on measured DCtransepithelial potential, impedance or other electrophysiologicalcharacteristic. The electrodes in the described methods and apparatuscan be used in contact with, in proximity to, over, or inserted into thetissues being examined. It should be understood that where the method isdescribed in an embodiment as encompassing one of these arrangements, itis contemplated that it can also be used interchangeably with the other.For example, where the method is described as having an electrode incontact with the tissue, the method can also be used with the electrodeinserted into or in proximity to the tissue. Similarly, where the methodis described as having an electrode in proximity to the tissue, it iscontemplated that the electrode can also be in contact with or insertedinto the tissue.

In order to more accurately detect transport alterations in abnormalpre-cancerous or cancerous epithelial tissue, a pharmacological agentmay be introduced to manipulate the tissue. Pharmacological agents mayinclude agonists of specific ion transport and electrical activity,antagonists of specific ion transport and electrical activity, ionicsubstitutions, and/or hormonal or growth factor stimulation orinhibition of electrical activity.

Depending on the location of the tissue to be investigated, a number ofmethods may be used to administer the pharmacological or hormonalagents. One exemplary method includes introducing the agent directly tothe tissue being investigated, via ductal infusion, perfusion, directcontact or injection. Another exemplary method includes applying theagent to the skin surface, wherein the agent acts transcutaneously, orthrough the skin. Yet another exemplary method includes electroporation,wherein the ductal epithelium or surface is made permeable by thepassage of alternating current via electrodes in contact or penetratingthe organ or epithelium of interest. The agent then passive diffusesinto the organ and its constituent cells. The agent may be introduceddirectly into the breast ductal system using the modified nippleaspirator cup and electrode, or lavaged into a specific duct using aductal catheter or probe. Additional exemplary methods include viainhalation, oral administration, lavage, gavage, enema, parenteralinjection into a vein or artery, sublingually or via the buccal mucosa,or via intraperitoneal administration. One skilled in the art willappreciate that other methods are possible and that the method chosen isdetermined by the tissue to be investigated.

Thus, systems and methods consistent with the present invention usetransepithelial electropotential or/and impedance measurements todiagnose pre-malignancy or cancer. Further, systems and methodsconsistent with the present invention use a defined set of frequencies,in combination, to characterize functional and structural alterations inpre-malignancy and cancer. By using spaced electrodes the presentinvention may provide topographic and geometrical (depth) informationabout the epithelium under examination to diagnose pre-malignancy andcancer. In one embodiment, systems and methods of the present inventionuse electrodes with specially formulated ECMs to provide functionalinformation about the epithelium to diagnose pre-malignancy and cancer.

In order to measure the transepithelial breast DC potential it isnecessary that the lumen of the duct be electrically accessed by anipple electrode constructed to make an electrical connection betweenthe Ag/AgCl (or similar low offset platinum/hydrogen, titanium, tin-leadalloy, nickel, aluminum, zinc, carbon, or other conductive metal orconductive polymer electrode) pellet recessed within the nipple cup. Thecup is filled with an ECM (electro-conductive medium), which enters theductal system passively, or after aspiration with a syringe or pump,making contact with the ductal lumen. A surface electrode placed at thesurface of the breast completes the electrical circuit, so thatmeasurements of transepithelial potential may be made between the ductalepithelium, or center of the tumor and the skin surface. Similarconsiderations have to be given to measure transepithelial AC impedancewhereby the measuring electrodes measure the voltage drop and phaseshift across the ductal epithelium or tumor, by utilizing a nippleelectrode in combination with a skin surface electrode. Otherconfigurations of this approach are more invasive, whereby measurementcan be made between an electrode inserted via a ductoscope or nippleduct probe electrode referenced to the skin or an IV (intravenous),intradermal, or subcutaneous electrode. In another embodiment, the ductmay also be accessed by a needle-electrode inserted through the skin.

In order to combine DC transepithelial measurement with impedancemeasurements, it is necessary to obtain baseline measurement of the DCpotential using the voltage sensing electrodes, referenced to surfaceelectrode with low-contact impedance, or the blood stream via an IV, orthe interstitial body fluid via a needle electrode or electrode thatpermeabilizes the overlying epidermis or other epithelium, or other bodyreference point. The electrodes may contain different ionicconcentrations, pharmacological agents or hormones in their ECMs. Asused in this description, an ECM is a medium that permits transmissionof electrical signals between the surface being measured and theelectrode. An agent includes any ionic concentration, pharmacologicalagent, hormone or other compound added to the ECM or otherwiseintroduced to the tissue under investigation, selected to providefurther information about the condition of the tissue. In anotherembodiment the concentrations of agents may be changed using a flowthrough system.

Electroconductive media can include conductive fluids, creams or gelsused with external or internal electrodes to reduce the impedance(resistance to alternating current) of the contact between the electrodesurface and the skin or epithelial surface. In the case of DC electrodesit is also desirable that the ECM results in the lowest DC offset at theelectrode surface, or an offset that can be measured. The ECM will oftencontain a hydrogel that will draw fluid and electrolytes from deeperlayers of the skin to establish electrical contact with the surfaceelectrode. Electrodes that are used to pass current require ECMs withhigh conductance. Usually this is accomplished by using ECMs with highelectrolyte content. The electrolytes frequently used are KCl (potassiumchloride) because of the similar ionic mobility of these two ions infree solution, so that electrode polarization is less of a problem thanwhen ions of different mobility are used. Other ions such as sodium maybe used in ECM formulations, and the higher electrolyte concentrationresult in more rapid electrode equilibration.

In situations where estimations will be made of the permeability of theepithelium to specific ions, the concentration of K in the ECM will bevaried so that the conductance of the epithelium to potassium may bemeasured electrophysiologically. An enhancer or permeant may be added tothe ECM to increase the conductance of the underlying skin to theelectrolyte in the ECM. Other approaches include mild surface abrasionwith pumice and alcohol to reduce surface skin resistance, abrasive padssuch as Kendall Excel electrode release liner (Tyco Health Care,Mansfield, Mass.), 3M Red Dot Trace Prep (3M Corporation, St. Paul,Minn.), cleaning the skin with alcohol, an automated skin abrasionpreparation device that spins a disposable electrode to abrade the skin(QuickPrep system, Quinton, Inc., Bothell, Wash.), ultrasound skinpermeation technology (SonoPrep, Sontra Medical Corporation, Franklin,Mass.), or silicon electrodes, which just penetrate the stratum corneumto reduce skin surface resistance. (See also, the comparison anddiscussion of several methods in Biomedical Instrumentation &Technology, 2006; 39: 72-77; the content of which is incorporated hereinby reference.)

In order to measure the depth of the impedance alteration, the voltagedrop will be made between surface electrodes with different spacing.Spacing will be determined by knowledge of the depth to be probed.Similarly two different frequency ranges will be used to measurefunctional and structural changes at different depths.

In order to more accurately detect the functional transport alterationsat different depths in abnormal pre-cancerous or cancerous epithelialtissue, a pharmacological agent is introduced to manipulate the tissue,while electrically probing the tissue at different frequencies andmonitoring the voltage drop between differently spaced electrodes.Pharmacological agents include agonists of specific ion transport andelectrical activity, antagonists of specific ion transport andelectrical activity, ionic substitutions, and/or hormonal or growthfactor stimulation or inhibition of electrical activity.

Depending on the location of the tissue to be investigated, a number ofmethods are used to administer the pharmacological or hormonal agents.One exemplary method includes introducing the agent directly to thetissue being investigated, via ductal perfusion, infusion, directcontact or injection. Another exemplary method includes applying theagent to the skin surface, wherein the agent acts transcutaneously, orthrough the skin. Yet another exemplary method includes electroporation,wherein the epithelium or surface is made permeable by the passage ofalternating current via electrodes in contact or penetrating the surfaceof the breast or ductal epithelium of interest. The agent then passivelydiffuses into the breast and its constituent cells. Additional exemplarymethods include via inhalation, oral administration, lavage, gavage,enema, parenteral injection into a vein or artery, sublingually or viathe buccal mucosa, or via intraperitoneal administration. One skilled inthe art will appreciate that other methods are possible and that themethod chosen is determined by the tissue to be investigated.

Based on the agent introduced and the tissue being investigated,measurements of electrophysiological properties, such as impedance, areperformed. Other properties that can be measured includes,transepithelial potential, changes in spontaneous oscillations intransepithelial potential or impedance associated with the malignantstate, time delay in a propagation signal between electrodes, whichindicates a loss of gap-junction function. If adjacent cells areelectrically coupled, one can examine the loss of coupling bypharmacologically eliciting an electrical signal and measuring thesignal propagation up and down-stream through surface epithelial cells.This is a functional measurement of the gap-junctions, whereas simpleelectrical stimulation will measure shunting of a current between thecells (a structural measurement, at least in the high frequency range).

The results of these measurements are then used to determine thecondition of the investigated tissue. For example, research hasindicated that specific ion transport processes are altered during thedevelopment of cancer. For example, a loss of electrogenic Na⁺transport, an up-regulation in Na/H exchange, a down-regulation in K⁺conductance, a decrease in basal Cl⁻ absorption, and a down-regulationin c-AMP (cyclic adenosine-3′,5′-cyclic monophosphate) stimulated Cl⁻secretion have been observed.

Thus, by administering agents appropriate to the particular epithelialtissue and measuring the associated electrophysiologicalcharacteristics, it is possible to detect abnormal pre-cancerous orcancerous tissue while the development of such tissue is at an earlystage. It should be understood that the method and system of the presentinvention is applicable to any epithelial derived cancer, such as, butnot limited to, prostate, colon, breast, esophageal, and nasopharyngealcancers, as well as other epithelial malignancies, such as lung,gastric, uterine cervix, endometrial, skin and bladder.

Specifically, in cancers affecting mucosal or epithelial tissues,transport alterations may be sufficiently large to suggest that they area consequence of an early mutation, affecting a large number of cells(i.e., a field defect). In this case, they may be exploited as potentialbiomarkers for determining which patients should be either morefrequently monitored, or conversely, may be used to identify particularregions of epithelium that require biopsy. The latter is especiallyhelpful in the case of atypical ductal hyperplasia or ductal carcinomain situ (DCIS), which are more difficult to detect mammographically, orby clinical breast examination without having to resort to an invasivebiopsy.

Applying the methods of the present invention, several observations havebeen made:

(1) Differences in the total impedance are observed when comparingmalignant breasts with benign or normal breasts. The total impedance ishigher comparing malignant to benign breasts with the total impedanceexceeding 50,000 ohms, or even higher, for the malignant breasts.

(2) Total capacitance was lower overall, comparing malignant with benignor normal breasts.

(3) The impedance curves for normal and malignant breasts separate atlower frequencies.

(4) The shape of the curves differs depending on the pathologicalcondition of the breast.

(5) Electrical resistance of the tumor may be lower at lowerfrequencies, for example, in the range of about 1 to about 0.1 hertz.This also depends on the type and size of the tumor.

(6) Capacitance of the tumor may be higher at the lower frequencies.This also depends on the pathological type and size of the tumor.

(7) Differences exist between phase angle, characteristic capacitanceand the suppression of the center of the impedance arc depending on thepathological status of the breast.

(8) When current is passed across a malignant tumor from another site onthe breast or body, rather than from the nipple to the surface of thebreast, the impedance may be lower when the voltage drop is measuredacross the tumor rather than between the nipple and the tumor i.e.,across ductal epithelium. The capacitance is usually higher when themeasurement is made across a malignant tumor, rather than across theductal epithelium. Therefore a combination of measurements; nipple tobreast surface (transepithelial impedance spectroscopy), body surface tobreast surface (transtumor impedance), and transepithelial potential(ductal epithelium in series with skin provides the optimum diagnosticinformation.

The methods of the present invention are particularly useful when use ismade of the entire frequency range between about 60,000 Hertz and about0.1 Hertz, although most of the discriminatory information is observedat frequencies below about 200 Hz. The preferred protocol is to take5-10 electrical measurements (impedance, reactance, phase angle,resistance, etc.) between about 60,000 Hz and about 200 Hz and then takeas many measurements as possible (taking into consideration, forexample, the comfort of the patient, response time of the equipment,etc.) between about 200 and about 0.1 Hz; preferably between about 150Hz and about 0.1 Hz; more preferably about 100 Hz and about 0.1 Hz; forexample between about 50 Hz and about 0.1 Hz. In practice, this can meantaking about 20 to about 40 measurements in one or more of the lowerfrequencies ranges.

It has also been observed that the application of alternating suctionand release opens up the nipple ducts so that impedances are generallylower if this protocol is followed. This will typically lower theimpedance in the high frequency range. This reduces measurement noiseand enhances current passage along the ducts to the tumor site. Furtherimprovement can be made in lowering the impedance of the nipple andlarger collecting ducts by using alcohol or a dekeratinizing agent suchas Nuprep® to remove keratin plugs in the surface duct openings on thenipple surface. Methods for opening up the ductal system are known forductoscopy (e.g., Acueity), obtaining nipple aspirate fluid (NAF) orductal lavage (e.g., CYTYC), but this technology has not previously beenapplied to the field of the present invention. A particularly improveddevice will employ an automated suction pump connected to a manometer tosuction rhythmically, analogous to a breast pump, then employ a holdingsuction pressure at a predetermined level and then change the holdingpressure to another level so that the effect of altered suction on theimpedance spectra can be used as a diagnostic test. Without wishing tobe bound by theory, it is believed that a difference in electricalresponse, for example a different impedance curve, arises due ductalcollapse with the application of suction in a normal breast whereas thepresence of malignancy in a duct inhibits such collapse.

Mechanical pressure can also be used to provide additional diagnosticinformation during DC and AC impedance measurement to characterizebreast tissue. In this manner, positive pressure is applied to the skinsurface and negative pressure, or vacuum is applied to the nipple. Inthis manner an additional approach can be used to obtain furtherdiagnostic information that can be independently used or can be used incombination with the technique relating to nipple aspiration.

Two patterns of impedance and open circuit potential have been observedfrom transepithelial impedance spectroscopy and DC measurements inpatients with benign or malignant breast lesions. FIG. 22 demonstratesone source of false positives that can occur with a low impedancefibroadenoma (a benign lesion). Two patterns exist in the impedancespectral profile of breast cancer. The first change is an increase inimpedance, particularly at low frequency. Without wishing to be bound bytheory, this is believed due to the ducts becoming packed with tumorcells (ductal-carcinoma in-situ, DCIS) which increases the resistance ofthe ductal epithelium. Once an invasive carcinoma and mass lesiondevelops within a duct system, the tight-junctions between cells breakdown, resulting in a decrease in impedance, particularly at lowfrequencies. In FIG. 22 the open circles demonstrate the impedancespectra of a control ductal system in a patient with a carcinoma of thebreast in a quadrant of the breast that is uninvolved by tumor. It canbe seen that the open circles on the right side of the graph form asecond circular arc (Cole plot) that extends beyond the right sidevertical Y-axis. This indicates that a high impedance ductal systemexists in the control quadrant of this patient's breast. In the oppositequadrant of the same patient's breast a mass has developed. Theimpedance spectrum of that breast quadrant is depicted by the opensquares. The second semicircular arc has now been replaced by a lowimpedance Cole plot that passes below the X-axis. Since we havepreviously observed that the low frequency impedance arc appears to bedominated by the terminal ducts, it is likely that the terminal ductshave become less, electrically resistant in the region of the developingcancer. This suggests a lower electrical impedance at this stage in thedevelopment of the cancer.

In another patient the impedance spectra over a suspicious mass appearssimilar to the developing cancer. This lesion had an impedance spectradepicted by filled squares. The impedance curve has lost its lowimpedance curve similar to the developing cancer. The patient underwenta biopsy, the results of which demonstrated that the mass was a benignfibroadenoma. Several features do however distinguish the developingcancer from the fibroadenoma:

(1). The middle part of the impedance arc is flattened in thefibroadenoma (filled squares) compared with the carcinoma (opensquares);

(2) The notch frequency occurs at about 30 Hz for cancer (this may occuras low as 1 Hz in cancer) and about 100 Hz for the fibroadenoma. Notchfrequency is the frequency at which there appear two separate RCtime-constants in the impedance spectra resulting in two incompletelyfused arcs. It is the frequency at which the two arcs or double humpsappear to partly separate. (See Jossinet et al., Ann. NY Acad. Sci.1999; 873: 30-41, incorporated herein by reference.) The acronym RCstands for Resistor-Capacitor. The product RC is referred to in the artas the time constant, and is a characteristic quantity of an RC circuit.For example, when t=RC, the capacitor has charged to a fraction equal to1-1/e, or about 63% of its final value. Typically the units of RC areseconds or milliseconds. When the time constant of the high frequency RCcomponents of the circuit have a significantly different time constantcompared to the low frequency RC components, the resulting figureexhibits two separate semi-circles on a Nyquist plot. If the timeconstants are close to one another, the semi-circles will appear to befused. With better separation, in other words time constants that differmore, the information obtained is more diagnostically useful. Asdescribed above, a greater degree of diagnostic information in thepresent invention is obtained in the low frequency range; and

(3) The subepithelial resistance (the intercept of the high-frequencyimpedance curve with the x-axis on the left side of the curve) is muchlower in the fibroadenoma (140 ohms) than in the cancer (420 ohms).

As noted above, an alternative approach that can be used to identifyabnormalities in the breast and distinguish benign from malignantdisease involves the application of mechanical pressure or compressionto occlude the ductal pathway and thereby increase the impedance pathwayfor the passage of electrical current through the breast. Theapplication of mechanical pressure or compression, in other wordspositive pressure (in contrast with the application of vacuum to, e.g.,nipple ducts as described elsewhere herein), can be accomplished byvarious means well-known to the skilled practitioner. For example, oneor more fingers or the hand can be used to palpate an area of thebreast, including a suspicious area or an adjoining area thereto.Alternatively, a mechanical device, such as a pressure transducer, canbe used to apply a finite or defined degree of pressure to a specificarea. Additionally, the use of mechanical pressure can be combined withthe use of suction or vacuum as described above. For convenience, theuse of mechanical pressure can be referred to as the mechanical pressureprotocol and it can be accomplished by one or more of the followingsteps in the suggested order or in other sequences:

(1) Measurement of an impedance spectrum over a region of tissue wherean abnormality is suspected (suspicious region).

(2) Measurement of an impedance spectrum over a region of tissue whereno abnormality is suspected (control region).

(3) The application of mechanical pressure or compression over thesuspicious region.

(4) The measurement of the impedance spectrum in the suspicious regionfollowing the application of mechanical pressure.

(5) The application of mechanical pressure or compression over thecontrol region.

(6) The comparison of the impedance profile before and after compressionin the suspicious region.

(7) The comparison of the impedance profile before and after compressionin the control region.

(8) The comparison of the impedance profile of the suspicious region tothe control region.

(9) Using 6, 7 and 8 alone or in combination to diagnose the normal ordiseased state of the tissue.

(10) Measurement of the kinetics of the change in impedance whenpressure is applied or released over a region of tissue.

(11) Using the kinetics of the change in impedance to diagnose thenormal or diseased state of the tissue.

(12) Using a combination of a single or multiple pressure transducerwith steps 3-11 to obtain both a pressure profile, and an impedanceprofile.

(13) Using a combination of the applied pressure profile with thechanges in the impedance profile to diagnose the normal or diseasedstate of the tissue.

(14) Using a combination of the 1-13 with changes in the suctionpressure applied to the nipple-sensor aspirator-cup.

(15) Using a combination of the altered impedance profile followingchanges in the applied suction pressure to the nipple-sensoraspirator-cup with steps 1-13 to diagnose the normal or diseased stateof the tissue.

Ducts containing tumor will generally be less compliant, and thereforeless compressible than normal ducts not filled with tumor cell. This isdepicted in FIG. 23. The open squares depict the impedance spectra of anormal duct which appears to have at least two time constants. The notchfrequency (the point at which the two impedance curves incompletelyfuse) in this normal duct is at about 4 Hz. When a pressure of up to 1kg cm⁻² was applied over the breast surface electrode a new impedancespectrum was measured at 59 frequencies logarithmically spaced between60,000 Hz and 0.1 Hz (filled squares). The impedance of the lowfrequency curve (below 4 Hz) is markedly increased due to occlusion ofthe more compliant duct. In contrast, there is virtually no effect onthe impedance curve above 4 Hz. FIG. 24 demonstrates the effect on alarge scale of the X-axis. Note that the impedance increases fromapproximately 7850 ohms to almost 42,060 ohms. The open squares (nocompression) are obscured by the closed squares (compression).

FIG. 25 depicts the release of compression on the duct (open circles)with the return of the impedance profile almost to control(pre-compression) levels. Note that the kinetics and shape of therelease curve has specific characteristics in normal as opposed toabnormal tissue. For example, the return of the impedance curve takesseveral minutes due to the compliance properties of the ductal andsurrounding parenchymal tissue. These properties can be used tocharacterize the pathological state of the tissue.

FIG. 26 demonstrates the same protocol applied to a cyst. The opensquare QOI2:00 (quadrant of interest at the 2:00 o'clock position)depicts the impedance spectrum over a cyst. When pressure is applied(closed square) the impedance decreases, as may be expected as thecurrent pathway is decreased because of a decrease in theanterior-posterior diameter of the cyst with compression. The controlquadrant is depicted by open circles, which pass off the scale. A largecyst is expected to have a lower impedance than the surrounding tissuebecause it conducts electricity better.

FIG. 27 demonstrates the same protocol applied to a region offibrocystic disease. Since the cystic component is minimal i.e., thereis a more non-compliant fibrous element in this example, there is aminimal effect observed with compression.

FIG. 28 demonstrates the same fibroadenoma shown in FIG. 22. Thisfibroadenoma has a significantly lower impedance than is usuallyobserved and the lesion can be confused with a carcinoma. The samepressure protocol was applied and an increase of impedance wasidentified although less than that observed in a normal duct (FIG. 24and FIG. 25). It should be noted that the surrounding ductal structureis less disrupted in a fibroadenoma than in carcinoma and therefore somecompression of the ductal structure was possible. The same compressionprotocol applied to the carcinoma in FIG. 22 results in no appreciablechange in the impedance profile, apparently because the ducts havealready been disrupted by the tumor and are less compressible.

FIG. 29 depicts a more typical fibroadenoma where the impedance ishigher. The control quadrant (open circles) has a somewhat noisyimpedance curve, but shows two partially fused RC curves because ofnormal ductal structure. In this case the pressure protocol results inminimal change in the impedance spectrum. As has been demonstrated, theapplication of pressure in combination with the electrical measurementsdescribed in detail above to selected regions of the breast exhibitingsuspicious tissue can be used effectively to distinguish betweenmalignant and other types of abnormal tissue.

A number of variations are possible for devices to be used with thepresent invention. Further, within a device design, there are a numberof aspects that may be varied. These variations, and others, aredescribed below.

One probe or other device includes a plurality of miniaturizedelectrodes in recessed wells. Disposable commercially available siliconchips processing functions, such as filtering, may perform surfacerecording and initial electronic processing. Each ECM solution or agentmay be specific to the individual electrode and reservoir on the chip.Thus, for one measurement, a particular set of electrodes is used. Foranother measurement, for example, at a different ionic concentration, adifferent set of electrodes is used. While this produces somevariations, as the electrodes for one measurement are not located at thesame points as for another, this system provides generally reliableresults.

An alternative approach is to use fewer electrodes and use aflow-through or microfluidic system to change solutions and agents.Specifically, solutions or agents are changed by passing small amountsof electrical current to move solution or agent through channels and outthrough pores in the surface of the probe. In this embodiment, theelectrode remains in contact with the same region of the skin or ductalepithelium, thus eliminating region-to-region variation in measurement.This approach requires time for equilibration between differentsolutions.

In detecting the presence of abnormal pre-cancerous or cancerous breasttissue, a hand-held probe is provided for obtaining surface measurementsat the skin. The probe may include electrodes for passing current aswell as for measuring. An impedance measurement may be taken between thenipple cup electrode and the hand-held probe, or may be taken betweenelectrodes on the hand-held probe. Alternatively, a ductoscopic ornon-optical ductal probe may be interfaced with one or more miniaturizedelectrodes. After taking initial DC measurements, awetting/permeabilizing agent may be introduced to reduce skin impedanceor one of the methods described hereinabove may be used. The agent maybe introduced using a microfluidic approach, as described above, to movefluid to the surface of the electrodes. Alternatively, surfaceelectrodes that just penetrate the stratum corneum may be used todecrease impedance.

Regardless of the configuration of the device, FIG. 1 is a schematic ofa DC and AC impedance measurement system 100 used in cancer diagnosis,consistent with the present invention. The system 100 interfaces with aprobe device 105 including multiple electrodes, wherein the actualimplementation of the probe device 105 depends on the organ andcondition under test. The probe device 105 may incorporate theelectrodes attached to a needle, body cavity, ductoscopic, non-opticalductal or surface probe. A reference probe 110 may take the form of anintravenous probe, skin surface probe, nipple-cup or ductal epithelialsurface reference probe depending on the test situation and region ofbreast under investigation.

To avoid stray capacitances, the electrodes may be connected viashielded wires to a selection switch 120 which may select a specificprobe 105 following a command from the Digital Signal Processor (DSP)130. The selection switch 120 also selects the appropriate filterinterfaced to the probe 105, such that a low pass filter is used duringDC measurements and/or an intermediate or high pass filter is usedduring the AC impedance measurements. The selection switch 120 passesthe current to an amplifier array 140 which may be comprised of multipleamplifiers or switch the signals from different electrodes through thesame amplifiers when multiple electrodes are employed. In a preferredembodiment digital or analogue lock-in amplifiers are used to detectminute signals buried in noise. This enables the measurement of thesignal of interest as an amplitude modulation on a reference frequency.The switching element may average, sample, or select the signal ofinterest depending on the context of the measurement. This processing ofthe signal will be controlled by the DSP following commands from theCPU. The signals then pass to a multiplexer 150, and are serializedbefore conversion from an analogue to a digital signal by the ADC. Aprogrammable gain amplifier 160 matches the input signal to the range ofthe ADC 170. The output of the ADC 170 passes to the DSP 130. The DSP130 processes the information to calculate the DC potential and itspattern on the ductal-epithelial or skin surface as well as over theregion of suspicion. In addition the impedance at varying depth andresponse of the DC potential and impedance to different ECMconcentrations of ions, drug, hormones, or other agent are used toestimate the probability of cancer. The results are then sent to the CPU180 to give a test result 185.

Alternatively the signal interpretation may partly or completely takeplace in the CPU 180. An arbitrary waveform generator 190 or sine wavefrequency generator will be used to send a composite waveform signal tothe probe electrodes and tissue under test. The measured signal response(in the case of the composite wave form stimulus) may be deconvolvedusing FFT (Fast Fourier Transforms) in the DSP 130 or CPU 180 from whichthe impedance profile is measured under the different test conditions.An internal calibration reference 195 is used for internal calibrationof the system for impedance measurements. DC calibration may beperformed externally, calibrating the probe being utilized against anexternal reference electrolyte solution.

FIG. 2 includes a handheld probe 400, consistent with the presentinvention, which may be applied to the surface of the breast. The probemay include a handle 410. The probe 400 may be attached, either directlyor indirectly using, for example, wireless technology, to a measurementdevice 420. The probe 400 may be referenced to an intravenous electrode,a skin surface electrode, other ground, nipple electrode, or ductalprobe electrode within the duct or at the nipple orifice. In oneembodiment, illustrated in FIG. 2, the reference is a nipple electrodeor ductal probe 430, illustrated in greater detail at close-up 440. Oneadvantage of this configuration is that DC electropotential andimpedance can be measured between the nipple electrode 430 and the probe400. The measurement is thus a combination of the DC potentials or/andimpedance of the breast ductal epithelium, non-ductal breast-parenchyma,and the skin.

Referring to close-up 440, the ductal probe is inserted into one ofseveral ductal orifices that open onto the surface of the nipple. Ductalprobe 443 is shown within a ductal sinus 444, which drains a largercollecting duct 445.

Another advantage of using a nipple electrode is that a solution forirrigating the ductal system may be exchanged through the probe,permitting introduction of pharmacological and/or hormonal agents. Asshown in magnified nipple probe 443, 443′ fluid can be exchanged througha side port. Fluid may be infused into the duct and aspirated at theproximal end (away from the nipple) of the nipple probe. Differentelectrolyte solutions may be infused into the duct to measure alteredpermeability of the ductal epithelium to specific ions or the epitheliummay be probed with different drugs to identify regions of abnormality.Estradiol, or other hormonal agents, may be infused into a breast ductto measure the abnormal electrical response associated withpre-malignant or malignant changes in the epithelium.

It should be understood that different configurations may also be used,such as a modified Sartorius cup that applies suction to the nipple.With this configuration, gentle suction is applied to a cup placed overthe nipple. Small amounts of fluid within the large ducts and ductsinuses make contact with the electrolyte solution within the Sartoriuscup, establishing electrical contact with the fluid filling the breastducts. DC or AC measurements may then be made between the cup and asurface breast probe.

FIG. 3 illustrates the probe 400 of FIG. 2 in greater detail. The skincontact of the surface 450 is placed in contact with the breast. Thesurface electrodes 451 measure DC or AC voltages. The current passingelectrodes 452 are used for impedance measurements. Probe 400 may alsoinclude one or more recessed wells containing one or more ECMs. Multiplesensor electrode arrays may be attached to the surface probe togetherwith current passing electrodes. The individual electrodes may berecessed and ECMs with different composition may be used topharmacologically, electrophysiologically, or hormonally probe thedeeper tissues or epithelium under test. Spacing of the electrodes maybe greater for the breast configuration than for other organ systems sothat deeper tissue may be electrically probed and the impedance of thedeeper tissue evaluated. This probe may either be placed passively incontact with the surface of the breast or held in place by pneumaticsuction over the region of interest. Ports may be placed for theexchange of solutions or for fluid exchange and suction (not shown).Guard rings (not shown) may be incorporated to prevent cross-talkbetween electrodes and to force current from the contact surface intothe breast. In this configuration there are four current passingelectrodes [453] each positioned radially 90° apart. This permitscurrent to be passed and the voltage response to be measured inperpendicular fields. The electrodes will be interfaced via electricalwire, or wireless technology, with the device described in FIG. 1 above.

Further embodiments of this technique may involve the use of spacedelectrodes to probe different depths of the breast, and the use ofhormones, drugs, and other agents to differentially alter the impedanceand transepithelial potential from benign and malignant breast tissue,measured at the skin surface. This enables further improvements indiagnostic accuracy.

FIG. 4 illustrates a nipple cup electrode [500] that may be used as areference, current passing, voltage measuring or combination electrode[502]. In this configuration suction and fluid exchange is applied tothe electrode housing [501] through a side port [510] connected by aflexible hose [515] to a suction device, aspirator or syringe (notshown). The flange [503] at the base of the cup is applied to the areolaof the breast [520]. Pneumatic suction is applied through the side portand communicated to the housing by passage [512] so as to obtain a sealbetween the breast [520] and the nipple electrode [501]. Electrolytesolution is used to fill the cup and make electrical contact with theunderlying ductal system. Fluid may be exchanged, or pharmacological andhormonal agents introduced, by applying alternating suction andinjecting fluid or drugs into the cup through the side port. Thepneumatic suction will open up the duct openings [505] either by itselfor after preparation with alcohol or de-keratinizing agents to removekeratin plugs at the duct openings at the surface of the nipple. Thenipple cup electrode [502] may be interfaced by means of an electricalconnection [530] or by a wireless connection (not shown) with thedevices illustrated in FIGS. 1-3 to obtain DC potential, AC impedance orcombination measurements.

FIG. 5 illustrates an alternative approach where an individual duct isprobed with a flexible catheter electrode [550] attached to a syringe[555]. This may be used when a specific duct produces fluid anddiagnosis is to be performed on the specific ductal system producing thefluid. In this configuration a saline filled syringe is connected to aflexible electrode [550], which is inserted into the duct

Fluid may be exchanged, or drugs and hormones may be infused into theduct, through the catheter. An electrode within, or attached to thesyringe makes electrical contact with the individual ductal system, andthe surface probe electrodes [552] complete the circuit so that the DCpotential, AC impedance or a combination of both may be measured acrossthe ductal epithelium, skin and intervening breast parenchyma incombination with the systems described in FIGS. 1-3. Another approachwould be to use a ductoscope in combination with a surface probe withthe electrode(s) interfaced with the ductoscope.

Devices to measure the electrophysiological characteristics of tissueand the differences between normal and abnormal tissue may include thoseknown in the art such as electrical meters, digital signal processors,volt meters, oscillators, signal processors, potentiometers, or anyother device used to measure voltage, conductance, resistance orimpedance.

DC potential is usually measured using a voltmeter, consisting of agalvanometer in series with a high resistance, and two electrodes (oneworking and one reference). Voltmeters may be analog or digital. Ideallythese should have an extremely high input resistance to avoidcurrent-draw. DC potential may also be measured with an oscilloscope.

Impedance may be measured using a number of approaches. Withoutlimitation, examples include phase-lock amplifiers, which may be eitherdigital or analog lock-in amplifiers. Pre-amplifiers may be used inconjunction with the lock-in amplifier to minimize stray currents toground improving accuracy. Digital lock-in amplifiers are based on themultiplication of two sine waves, one being the signal carrying theamplitude-modulated information of interest, and the other being areference signal with a specific frequency and phase. A signal generatorcan be used to produce the sine waves or composite signal to stimulatethe tissue. Analog lock-in amplifiers contain a synchronous rectifierthat includes a phase-sensitive detector (PSD) and a low-pass filter.Other approaches include the use of an impedance bridge with anoscillator to produce an AC sine wave. These devices when automated arereferred to as LCR-meters and use an auto-balancing bridge technique.Constant current or constant voltage current sources may be used. In onepreferred embodiment, a constant current source is used. Rather than anoscillator with a fixed frequency signal a signal generator, whichproduces, superimposed sine waves may be used.

The tissue response is deconvolved using fast Fourier transforms orother techniques. Bipolar, tripolar or tetrapolar current and voltageelectrodes may be used to make measurements. In one preferred embodimenttetrapolar electrode configurations are employed to avoid inaccuraciesthat are introduced due to electrode polarization and electrode-tissueimpedance errors. Rather than impedance, current density may be measuredusing an array of electrodes at the epithelial or skin surface.Impedance may also be measured using electromagnetic induction withoutthe need for electrode contact with the skin or epithelium.

In order to process large amounts of data, the methods of the presentinvention could be implemented by software on computer readable mediumand executed by computerized equipment or central processor units.

Example 1 Breast Cancer

As mentioned above, impedance and DC electrical potential have been usedseparately at the skin's surface to diagnose breast cancer. Neither ofthese methods measures the ductal transepithelial DC or AC electricalproperties of the breast. This significantly reduces the accuracy of theapproach, because the origins of breast cancer are within the ductalepithelium, and not the surrounding breast stroma. Accuracy is furtherimproved when the transepithelial measurements of impedance and DCpotential are combined. The use of pharmacological and/or hormonalagents in combination with impedance or DC electrical potentialmeasurements, provide a more effective method for detecting abnormalpre-cancerous or cancerous breast tissue.

Breast cancer develops within a background of disordered proliferation,which primarily affects the terminal ductal lobular units (TDLUs). TheTDLUs are lined by epithelial cells, which maintain a TEP(transepithelial potential). In regions of up-regulated proliferation,the ducts are depolarized. The depolarization of ducts under the skinsurface results in skin depolarization. The depolarization issignificantly attenuated compared to that which is observed using atransepithelial ductal approach, as opposed to a non-transepithelialskin surface approach such as disclosed in U.S. Pat. Nos. 6,351,666;5,678,547; 4,955,383. When a tumor develops in a region of up-regulatedproliferation, the overlying breast skin becomes further depolarizedcompared with other regions of the breast and the impedance of thecancerous breast tissue decreases. The changes in ductal epithelialimpedance are not measured using existing technologies resulting in adiminution in accuracy. Alterations in TEP and impedance occur under theinfluence of hormones and menstrual cycle.

For example, the electrophysiological response of breast tissue to17-β-estradiol has been observed to be different in pre-cancerous orcancerous epithelium than in normal breast epithelium. In one method ofthe present invention, estradiol is introduced directly into the duct orsystemically following sublingual administration of 17-β-estradiol (4mg). This agent produces a rapid response, which peaks at approximately20 minutes. The electrophysiological response depends, in part, on thestage of the patient's menstrual cycle, as well as the condition of thebreast tissue. Specifically, in normal breast tissue, a rise in TEP willoccur during the follicular (or early) phase. In pre-cancerous orcancerous tissue, this response is abrogated. Post-menopausal women atrisk for breast cancer may have an exaggerated TEP response to estradiolbecause of up-regulated estrogen receptors on epithelial cell surfaces.

Furthermore, estrogen, progesterone, prolactin, corticosteroids,tamoxifen or metabolites, (all of which alter the ion transportcharacteristics of ductal epithelium depending on its premalignant,malignant and functional state), thereof may be introduced eitherorally, intravenously, transcutaneously, or by intraductal installation.

In one embodiment of the present invention, breast or other cancers maybe diagnosed by examining the basal conductance state of theparacellular pathway of the epithelium. For example, in the breast, asubstance known to affect the conductance of the tight junctions may beinfused into the duct, or administered by other mean, and thetransepithelial impedance and/or the DC potential of the breast ismeasured, before and after the administration of the agent, using acombination of surface, nipple, ductal or other electrodes. Thedifference in the transepithelial electrical response of the tightjunctions to the agent in normal compared to pre-malignant or malignantbreast epithelium is then is used to diagnose the presence or absence ofmalignancy.

In another embodiment, the electrodes are placed over the suspiciousregion and the passive DC potential is measured. Then AC impedancemeasurements are made as discussed below. The variable impedanceproperties of the overlying skin may attenuate or increase the measuredDC surface electropotentials. Alternatively, impedance measurements atdifferent frequencies may initially include a superimposed continuoussine wave on top of an applied DC voltage. Phase, DC voltage and ACvoltage will be measured. The resistance of the skin or other epitheliumat AC and a different resistance at DC are measured. Under DC conditionssince there is no phase shift, it is possible to measure thetransepithelial potential at the surface. The capacitive properties ofthe skin may allow the underlying breast epithelial and tumor potentialto be measured at the skin surface.

Once the ECM results in “wetting” of the skin surface there ispseudo-exponential decay in the skin surface potential using the abovereferenced approach. Ions in the ECM diffuse through the skin and makeit more conductive, particularly because of changes in the skin parallelresistance. The time constant for this decay is inversely proportionalto the concentration and ionic strength of the gel. Once the skin isrendered more conductive by the ECM the capacitive coupling of thesurface to the underlying potential of the tumor or the surroundingepithelium is lost so that the measured potential now reflects an offsetand diffusion potential at the electrode-ECM-skin interfaces.

FIG. 6 demonstrates the effect of varying the ionic content of thebathing Ringers solution on transepithelial conductance. The humanbreast epithelial cells were grown as monolayers on Millipore filtersand grew to confluence in 7 to 10 days. The epithelia were then mountedin modified Ussing chambers and the DC conductances were measured usinga voltage clamp. The conductance was measured by passing a 2 μA currentpulse for 200 milliseconds and measuring the DC voltage response andcalculating the transepithelial conductance (y-axis), and plotting itagainst time (x-axis). The conductance was measured first in standardRinger solution, then in a sodium-free Ringer, then returned to standardRinger, then in a potassium-free Ringer and finally returning tostandard Ringer solution while maintaining normal osmolality during thestudies.

The upper plot (filled squares and solid line) demonstrates theconductance of benign human breast epithelia grown as a monolayer. Theconductance is higher in the benign epithelial cells. The Na⁺ and K⁺components of conductance are approximately, 10 and 5 mS·cm⁻²respectively.

The lower plot (filled circles and dotted line) demonstrates theconductance of malignant human breast epithelia grown as a monolayer.The conductance is significantly lower in the malignant epithelialcells. The Na⁺ and K⁺ components of conductance are approximately, 4 and1 mS·cm² respectively.

In malignant tumors as opposed to monolayers of malignant epithelialcells, the tight junction between cells break down and the tumor becomesmore conductive than either benign or malignant epithelial monolayers.This observation may be exploited in the diagnosis of breast cancer. Thelower conductance of the epithelium around a developing tumor, togetherwith a region of high conductance at the site of the malignancy, may beused to more accurately diagnose breast cancer. Using electrodes withECMs with different ionic composition will permit the specific ionicconductances to be used in cancer diagnosis. For example a highconductance region with a surrounding area of low K-conductance isindicative of breast cancer; a high conductance area with a surroundingregion of normal conductance may be more indicative of fibrocysticdisease (a benign process).

FIG. 7 demonstrates measurements of cell membrane potential (ψ) in humanbreast epithelial cells. Measurements were made using a potentiometricfluorescent probe, and ratiometric measurements, which are calibratedusing valinomycin and [K⁺]-gradients. ψs were measured in the presence(closed circles) and absence (open circles) of estradiol (the activemetabolite of estrogen). Each symbol is the mean measurement. The uppererror bar is the standard error of the mean, and the lower error bar isthe 95% confidence level for the observations. The addition of estrogento cultured breast epithelial cells results in an instantaneous increasein ψ (data not shown) as well as the transepithelial potential see FIG.8. Transepithelial potential (V_(T)) of an epithelium is the sum of theapical (luminal) cell membrane potential (V_(A)) and the basolateral(abluminal) cell membrane potential (V_(BL)). ThereforeV_(T)=V_(A)+V_(BL) (changes in V_(A) and/or V_(BL) will therefore alterV_(T) or transepithelial potential).

FIG. 7 demonstrates that benign breast epithelial cells have a ψ ofapproximately −50 mV in the absence of estradiol and −70 mV whenestradiol is added to the culture media. Malignant and transformed cellshave a ψ of between −31 and −35 mV in the absence of estradiol andapproximately 50 mV when estradiol is present in the culture medium.

The difference in the electrical properties may be exploited to diagnosebreast cancer in vivo. Surface electropotential measurements are acombination of the transepithelial potential, tumor potential andoverlying skin potential. Physiological doses of estradiol may beadministered to the patient to increase ψ and the sustained effect ofestradiol results in an increase in transepithelial potential and tumorpotential measured as an increase in surface electropotential. Theincrease following sustained exposure (as opposed to the instantaneousresponse) is less in malignant than benign breast tissue.

It should be noted that the instantaneous response, illustrated in FIG.8, is greater in malignant epithelia, whereas the chronic or sustainedexposure to estradiol results in a lower increase in TEP(transepithelial electropotential) in malignant cells. Concurrentmeasurement of surface electropotential and impedance allow the moreaccurate diagnosis of cancer. FIG. 8 demonstrates the instantaneouseffect of increasing doses of estradiol on the transepithelial potential(TEP) of benign and malignant human breast epithelial cells. The cellswere grown as monolayers on Millipore filters and grew to confluence in7 to 10 days. The epithelia were then mounted in modified Ussingchambers and the TEP was measured using a voltage clamp. Increasingdoses of estradiol between 0 and 0.8 μM were added (x-axis). Thetransepithelial potential was measured after each addition and the TEPwas measured (y-axis).

The different dose response is apparent for benign and malignantepithelia. Malignant epithelia have a lower TEP but undergo aninstantaneous increase in TEP of approximately 9 mV (becomes moreelectronegative and reaches a level of <−6 mV) after exposure to only0.1 μM estradiol and then depolarize to approximately −2 mV withincreasing doses of estradiol up to about 0.5 μM. Benign epithelia havea lesser response to increasing doses of estradiol and do not peak untilalmost 0.3 μM and then remain persistently elevated (higher electronegativity), unlike the malignant epithelia, with increasing doses ofestradiol.

This difference in dose response may be exploited to diagnose breastcancer. Estradiol, or other estrogens, at a low dose will beadministered systemically, transcutaneously, intraductally, or by otherroute. The instantaneous response of the surface electropotential and/orimpedance may then be used to diagnose breast cancer with improvedaccuracy over existing diagnostic modalities using impedance or DCmeasurement alone.

FIG. 9 shows conductance measurements made at 2000 Hz at the surface ofthe breast. At this frequency the influence of the overlying skinimpedance is less. There is still however some variable component ofskin impedance, which results in significant variability of themeasurement as evidenced by the overlapping error bars. Each symbolrepresents the median measurement with error bars the standard deviationof the mean.

Open symbols represent measurements made in patients with a biopsyproven malignancy, while closed symbols represent measurements made inpatients whose subsequent biopsy proved to be a benign process such asfibrocystic disease. Malignant lesions are often associated withsurrounding breast epithelium that demonstrates Up-regulatedproliferation. These regions (“adjacent region”) are depolarized and mayhave a lower conductance than either over the region of malignancy. Thisdecreased conductance may be because of decreased K⁺-conductance of theadjacent and pre-malignant epithelium as I have observed in human colon.

Each of the three groups of symbols represents measurements from over asuspicious lesion or region, then the adjacent region, and then overnormal breast in an uninvolved quadrant of the breast. The first twosymbols (circles) in each of the three groups are impedance measurementswhere the median value is plotted against the left y-axis as conductancein mS·cm⁻². The second two symbols (squares) is the surface electricalpotential measured in mV and plotted against the right y-axis; eachdivision equals 5 mV. The third two symbols (triangles) are theelectrical index for benign and malignant lesions and are in arbitraryunits and are derived from the conductance and surface potentialmeasurement. It is immediately apparent that there is less overlap inthe error bars (standard deviation of the mean). Therefore breast cancercan be more accurately diagnosed using a combination of surfacepotential measurement and AC-impedance measurements. Furtherenhancements of this technique will involve the use of spaced electrodesto probe different depths of the breast, and the use of the hormones,drugs and other agents to differentially alter the impedance andtransepithelial potential from benign and malignant breast tissue, andmeasured at the skin or duct surface. This will enable furtherimprovements in diagnostic accuracy.

It should be understood that the surface potential measurement of breasttissue varies based on the position of the woman in her menstrual cycle.FIG. 10 illustrates this variance. This figure demonstrateselectropotential measurements taken over the surface of each breast at 8different locations with an array of 8 electrodes on each breastreferenced to an electrode on the skin of the upper abdomen.Measurements are taken with error bars equal to the standard error ofthe mean. Filled circles and filled squares represent the median valuefrom the left and right breast respectively. The vertical dotted line isthe first day of each menstrual cycle.

It can be seen that the median values for each breast tend to track oneanother with lower values in the first half of menstrual cycle(follicular phase) and higher values in the latter part of cycle (lutealphase). Although the measured electrical values are not completelysuperimposed, because of other factors affecting the electropotential ofthe breast, it can be seen that the lowest levels of electropotentialare observed 8-10 days before menstruation and the rise to the highestlevels around the time of menstruation. This may be because estradiollevels are higher in the second part of menstrual cycle and directlyaffect breast surface electropotential.

The cyclical pattern of electropotential activity when a breast canceror proliferative lesion is present is quite different. Similarly higherlevels of surface electropotential are observed when measurements weremade in the afternoon compared with the morning. This information can beexploited in a number of different ways. Measurement of the surfacepotential and impedance at different times during cycle enables a moreaccurate diagnosis because of a different cyclical change in surfaceelectropotential (i.e., the peak to peak change in potential is lessover a malignant region, relative to normal areas of the breast).Secondly, estradiol or another agent that changes the electropotentialof the breast may be administered systemically, topically (transdermal),intraductally or by other means, and the drug or hormone-induced changein surface potential may be used as a provocative test to diagnosebreast cancer.

FIG. 11 is a diagram illustrating the histological andelectrophysiological changes that occur during the development of breastcancer. The continuum from normal ductal epithelium, throughhyperplasia, atypical hyperplasia, ductal carcinoma in situ (DCIS), toinvasive breast cancer is thought to take 10 to 15 years. Some of thesteps may be skipped although usually a breast cancer develops within abackground of disordered ductal proliferation. The normal duct maintainsa transepithelial potential (inside of duct negatively charged), whichdepolarizes and impedance, which increases during the development ofcancer. Once an invasive breast cancer develops the impedance decreaseswith loss of tight junction integrity, and conductance through the tumoris enhanced. The disordered ducts have altered electrophysiogical andion transport properties. These properties are illustrated in the loweraspect of FIG. 11. These electrophysiological and transport alterationswill be exploited to diagnose cancer and premalignant changes in thebreast.

In these ways breast cancer can be more accurately diagnosed usingtransepithelial measurements of potential, or impedance, or acombination of transepithelial surface potential measurement,AC-impedance measurements and pharmacological manipulations.

Example 2 Chemopreventative and Therapeutic Use

In addition to the ionic, pharmacologic, and hormonal agents describedabove, the system and method of the present invention may be used withcancer preventative and therapeutic agents and treatments. Specifically,electrical measurement of altered structure and function provides amethod for evaluating a patient's response to the drugs withoutrequiring a biopsy and without waiting for the cancer to furtherdevelop. Patients who respond to a given chemopreventative ortherapeutic agent would likely show restoration of epithelial functionto a more normal state. Patients who do not respond would show minimalchange or may even demonstrate progression to a more advanced stage ofthe disease. This system and method, thus, may be used by eitherclinicians or drug companies in assessing drug response or by cliniciansin monitoring the progress of a patient's disease and treatment, ormonitoring the process of carcinogenesis (cancer development), before anovert malignancy has fully developed.

Example 3 Electrophysiological Changes in Other Epithelia

The examples illustrated by FIGS. 12 and 13 were performed in humancolon specimen removed at the time of surgery. Based on in vitro studiesin breast epithelial tissues, similar changes in human ductal epitheliumthat can be measured in vivo are expected.

FIG. 12 demonstrates the short circuit current (I_(SC)) of human colonicepithelium ex-vivo. The figure demonstrates the time course along thex-axis while varying the potassium gradient across the tissue. Thepotassium permeability of the apical membrane of human colonic mucosa(P^(K) _(a)) was determined in surgical specimens of controls andgrossly normal-appearing mucosa obtained 10-30 cm proximal to colorectaladenocarcinomas. The mucosa was mounted in Ussing chambers and thebasolateral membrane resistance and voltage were nullified by elevatingthe K⁺ in the serosal bathing solution. The apical sodium (Na⁺)conductance was blocked with 0.1 mM amiloride. This protocol reduces theequivalent circuit model of the epithelium to an apical membraneconductance and electromotive force in parallel with the paracellularpathway as has been verified by microelectrode studies. Increasingserosal K⁺ caused the I_(sc) to become negative (−140 μA/cm²) in normalcolon after which 30 mM mucosal TEA caused an abrupt increase in I_(sc)corresponding to block of apical K⁺ channels. In cancer-bearing colonthe reduction in I_(sc) is to −65 μA/cm². The serosal bath was remainedconstant at 125 mM [K].

FIG. 13 demonstrates that ΔI_(sc), determined with respect to the I_(sc)at 125 mM mucosal K, is a linear function of the concentration gradient,Δ[K]. Because the voltage across the apical membrane is zero under theseconditions and the paracellular pathway is nonselective, the P^(K) _(a)(apical potassium permeability) can be calculated using the Fickequation i.e., I_(sc)=F·P^(K) _(a)·Δ[K] where F is the Faraday constantand Δ[K] is the concentration difference for K⁺ across the epithelium.FIG. 13 demonstrates mean±sem values for I_(sc) in both normal andpremalignant human distal colon. The apical K⁺ permeability of controlswas 9.34×10⁻⁶ cm/sec and this was significantly reduced by 50% inpremalignant human mucosa to 4.45×10⁻⁶ cm/sec. P^(K) _(a) could also becalculated for the change in I_(sc) when the K⁺ channels were blockedwith TEA, assuming complete block. This resulted in somewhat lowervalues of 6.4×10⁻⁶ cm/sec and 3.8×10⁻⁶ cm/sec corresponding to a 40%reduction in P^(K) _(a).

These observations show that there is a field change in the K⁺permeability and conductance of human colon, during the development ofcancer. Similar results are expected in breast ductal epithelium.Impedance measurements, and/or DC measurement using electrodes withdifferent potassium gradients together with specific drugs, such asamiloride to block the contributions, of electrogenic Na⁺ transport; tothe electrical properties of the breast may be useful to diagnose breastcancer. Amiloride may be introduced through the breast duct and then theK⁺-concentration varied in the ECM used in the nipple electrode orirrigated into the duct to measure the reduced potassium permeabilityobserved in the surrounding breast ductal epithelium (with atypicalductal hyperplasia or early DCIS), or increased permeability in theregion of the developing invasive breast cancer.

FIG. 14 illustrates multiple Nyquist impedance plots from human breasts.Current was passed between a nipple cup electrode containing aphysiological saline solution under suction to open up the breast ductson the surface of the nipple, and an electrode placed on the surface ofthe breast. Voltage was then measured between the nipple and the regionof interest using a separate set of voltage measuring electrodes. Allmeasurements were made at 59 frequencies logarithmically spaced between60,000 hertz and 1 hertz except for the fibrocystic with atypia case(filled squares), which was measured at 59 frequencies between 60,000hertz and 0.1 hertz. The impedance curves demonstrate the lowestimpedance at highest frequencies. As the frequency of the applied sinewave decreases the curves shift from left to right along the x-axis.

FIG. 15 illustrates the impedance profile for a patient with ahemorrhagic cyst. These studies were performed at frequencies from60,000 hertz to 0.1 hertz. Measurements were made over the mass (lesion)in the 4 o'clock location of the breast and control measurements weremade in the 10 o'clock location of the same breast. The high frequencymeasurement demonstrates that the curves were superimposable. Separationbegins at a frequency below 5 Hz. The resistance of the mass was higherthan the control quadrant of the breast at low frequencies. Surface opencircuit potential measurements showed depolarization of only 2 mV overthe mass and therefore enabled discrimination from cancer despite thehigh impedance.

FIG. 16 illustrates a Bode plot of impedance plots comparing a patientwith fibrocystic disease (0465) and a patient with breast cancer((0099). It can be seen that the impedance [Z] and, theta (phase angle)separate at the lowest frequencies (open and closed symbols). The datafor the suspicious mass, which was identified as fibrocystic disease onpathology, the control region and the control region from the breastcancer are almost superimposable. At the low frequency end of thespectrum the cancer (0099D-filled circles) separates from the controlquadrant measurement (0099C-open circles) at approximately 20 Hz.

FIG. 17 illustrates the same data as in FIG. 16 plotted as a Nyquistplot. The mass (a region of fibrocystic disease 0465A-open squares) hasa 5000Ω lower impedance at the low frequency end of the curve to theright side of the x-axis, compared with the control region (0465B filledsquares). Plot 0099C (open circles) has a similar total impedance to thebreast with fibrocystic changes but the curve shows a “double hump”indicating two different time constants (τ) for the low and highfrequency ends of the impedance spectra in the malignant breast. Thischaracteristic appearance can also be utilized as a diagnostic tool.

FIG. 18 demonstrates the impedance spectra when the curve (0099D-filledcircles) is added for the breast cancer to FIG. 17. The total impedanceis significantly higher at 76397Ω compared to 45447Ω for the controlquadrant, and the lower (high frequency) curves begin to separate belowabout 200 Hz. The cancer was depolarized by 26 mV compared with thecontrol quadrant, and the fibrocystic disease was depolarized by 5.5 mV.The combination of higher impedance and greater depolarization enableddiagnosis of breast cancer in one patient and fibrocystic disease in theother despite the fibrocystic patient having impedances close to50,000Ω, a high value typically, but not necessarily suggesting thepresence of cancer.

FIG. 19 illustrates the effects of altering the level of suction appliedto the nipple cup electrode. Holding suction was established where 3 mlof saline was aspirated from the nipple cup and is illustrated as theimpedance curve with open squares. The impedance is approximately26,000Ω. When an additional 2-3 ml of saline is aspirated from thenipple cup electrode the impedance curve collapses to an impedance ofapproximately 3000Ω-filled squares. The addition of 1-2 ml of salineresulted in an increase in impedance (open circles), which increased toapproximately 15000Ω after 5 minutes (closed circles).

FIG. 20 illustrates similar suction pressure experiments on a malignantbreast: aspirating 3 ml of physiological saline from the nippleelectrode, after holding suction is obtained, results in a decrease intotal impedance from 45,447Ω to 29,029Ω in the control quadrant whereasimpedance decreases from 76,937Ω to 62,568Ω over the cancer. Thegreatest decrease in impedance is seen at the high frequency end of theimpedance spectra (curves on the left side of the X-axis). For example,the impedance decreases from 29216 to 1550Ω in the control quadrant, andfrom 35824 to 10106Ω over the cancer. On the other hand, the changes inimpedance are much less in the lower frequency spectra (curves on theright-side of the X-axis). A higher suction results in a decrease inimpedance from 19985 to 16593Ω in the control quadrant whereas thechange is even less over the cancer decreasing from 72674 to 71229Ω.Capacitance increases in the control quadrant, in the low frequencyrange when suction is increased from 1.50E-5 to 1.76E-5F (farads),decreased from 1.17E-5 to 9.32E-6F over the cancer. It can be seen thataltering the suction on the nipple cup electrode manually or with anautomated suction pump can be used to distinguish malignant from benignbreast epithelium by observing different responses of the impedancespectra to this maneuver.

FIG. 21 illustrates the method used for estimating the impedance for thecancer high suction curve in FIG. 20, where an arc is fitted to theimpedance data and extrapolated to the x-axis at each end of the curve.The difference between the low and high intercepts is the estimatedresistance of 72674Ω. The Capacitance (C) is estimated from thereactance at the maximum height of the arc, and is 1.1652E-5. TheDepression Angle (15.932) is the angle between the x-axis and a linedrawn from the origin of the x-axis to the center of the plotted arc.

DEVICES FOR USE WITH THE PRESENT INVENTION

A number of variations are possible for devices to be used with thepresent invention. Further, as noted above, within a device design,there are a number of aspects that may be varied. These variations, andothers, are described below.

One embodiment of a probe or other device for use in the presentinvention includes a plurality of miniaturized electrodes in recessedwells. Surface recording and initial electronic processing, such asfiltering, may be performed by disposable commercially-available siliconchips. Each ECM solution or agent may be specific to the individualelectrode and reservoir on the chip. Thus, for one measurement, aparticular set of electrodes would be used. For another measurement, forexample, at a different ionic concentration, a different set ofelectrodes would be used. While this produces some variations, as theelectrodes for one measurement are not located at the same points as foranother, this system provides generally reliable results.

An alternative approach is to use fewer electrodes and use aflow-through or microfluidic system to change solutions and drugs.Specifically, solutions or agents are changed by passing small amountsof electrical current to move solution or agent through channels and outthrough pores in the surface of the device. In this embodiment, theelectrode remains in contact with the same region of the surface of thebreast, thus eliminating region-to-region variation in measurement. Thisapproach requires time for equilibration between different solutions. Indetecting the presence of abnormal pre-cancerous or cancerous breasttissue, a hand-held probe is provided for obtaining surface measurementsat the skin. The probe may include electrodes for passing current aswell as for measuring. An impedance measurement may be taken between thenipple cup electrode and the hand-held probe, between a nipple cupelectrode and adhesive skin electrodes, between electrodes on aminiature ductoscope, between electrodes on a ductoscope and the skinsurface electrodes, or may be taken between electrodes on the hand-heldprobe. After taking initial DC measurements, a wetting/permeabilizingagent may be introduced to reduce skin impedance. The agent may beintroduced using a microfluidic approach, as described above, to movefluid to the surface of the electrodes. Alternatively, surfaceelectrodes that just penetrate the stratum corneum may be used todecrease impedance.

Fluids for use with the present inventions could include variouselectrolyte solutions such as physiologic saline (e.g. Ringers) with orwithout pharmacological agents one preferable electrolyte solution toinfuse into the ductal system will represent a physiological Ringersolution. Typically this consists of NaCl 6 g, KCl 0.075 g, CaCl₂ 0.1 g,NaHCO₃ 0.1 g, and smaller concentrations of sodium hyper andhypophosphate at a physiological pH of 7.4. Other electrolyte solutionmay be used were the electrolyte comprises approximately 1% of thevolume of the solute. Hypertonic or hypotonic solutions that are greateror less than 1% may be used in provocative testing of the epitheliumand/or tumor. The concentration of Na, K and Cl will be adjusted underdifferent conditions to evaluate the conductance and permeability of theepithelium. Different pharmacological agents such as amiloride (to blockelectrogenic sodium absorption), Forskolin (or similar drugs to raisecyclic-AMP) and hormones such as prolactin or estradiol can also beinfused with the Ringer solution to examine the electrophysiologicalresponse of the epithelium and tumor to these agents. Similarly, thecalcium concentration of the infusate will be varied to alter the tightjunction permeability and measure the electrophysiological response ofthe epithelium to this manipulation. Dexamethasone may be infused todecrease the permeability of the tight junctions, and theelectrophysiological response will be measured.

Although specific examples have been given of drugs and hormones thatmay be used in “challenge” testing of the epithelium and tumor, anyagonist or antagonist of specific ionic transport, or tight-junctionalintegrity, known to be affected during carcinogenesis may be used,particularly when it is known to influence the electrophysiologicalproperties of the epithelium or tumor.

Regardless of the configuration of the device, a signal is used tomeasure either the ductal transepithelial potential by itself, or thetransepithelial impedance. These two measurements may then be combinedto characterize the electrical properties of the epithelium associatedwith a developing abnormality of the breast, and are then compared withuninvolved areas of the same or opposite breast. Surfaceelectropotential measurements and impedance measurements are then madeto characterize the non-transepithelial electrical properties of thebreast. These measurements involve DC potential measurements where thesurface potential is referenced to an electrode that is not in contactdirectly, or indirectly through an ECM, with the duct lumen. Impedancemeasurements are similarly made between surface electrodes or a surfaceelectrode and a reference electrode not in contact directly orindirectly (through an ECM) with the ductal lumen. These measurementsare then compared and combined with the transepithelial electricalmeasurements to further characterize the breast tissue.

Furthermore an understanding of the electrophysiological basis of thealtered impedance or DC potential permits more accurate diagnosis. Forexample impedance or DC potential may increase or decrease because ofseveral factors. Increased stromal density of the breast may alter itsimpedance. This is a non-specific change, which may not have bearing onthe probability of malignancy. On the other hand, a decrease in thepotassium permeability of the epithelia around a developing malignancywould increase impedance and would be more likely associated with adeveloping cancer than a non-specific impedance change. Additionalinformation is obtained from my method by probing the tissue todifferent depths using spaced voltage-sensing electrodes. The use ofelectrophysiological, pharmacological and hormonal manipulations toalter DC potential and/or DC potential differentially in normal comparedto cancer-prone, pre-malignant or malignant tissue is anothersignificant difference, which enhances the diagnostic accuracy of myinvention over the above referenced ones.

Although the use of a nipple cup electrode has been described in thisapplication for use in breast cancer diagnosis, a cup electrode may beused in other organs where the epithelium may be difficult to accessendoscopically, or an endoscopic approach is not desired. An examplewould be the pancreatic and bile ducts, which join and open at theampulla of Vater within the second part of the duodenum. Bile ducttumors develop from the endothelial lining of the bile duct, (i.e.,cholangiocarcinomas, or the epithelial lining of the pancreatic duct,i.e., pancreatic carcinomas). The ampulla may be accessed endoscopicallyand a cup electrode applied by suction to the ampulla. Physiologicalsaline can be infused into the ducts and then a transepithelialpotential and impedance could be measured intraoperatively to identifythe region of tumor in the pancreatic, or bile duct using a secondelectrode placed on the peritoneal surface of the pancreas or bile duct.Alternatively, the peritoneal surface electrode may be replaced by askin surface, or intravenous electrode when used in a minimally ornon-invasive manner.

Drugs may be infused though the cup electrode as a provocative test anddescribed for breast. Secretin for example, stimulates bicarbonatesecretion by the pancreatic ducts. This response may be abrogated bychanges in the epithelium associated with pancreatic carcinoma. Thedistribution of muscarinic receptors, particularly M1 and M3, may bealtered in the epithelium during pancreatic carcinogenesis. Thereforespecific muscarinic agonists (cholinomimetic choline esters and naturalalkaloids) and antagonists (atropine, Pirenzepine (M1), Darifenacin(M3)) may be used to elicit a particular electrophysiological responsedue to chloride secretion in ductal epithelium associated withpancreatic cancer. Similar approaches may be used in the intra andextrahepatic bile ducts to diagnose liver cancer.

Prostatic cancer may be diagnosed using a urethral cup electrode appliedto the external urethral meatus. Physiological saline is infused intothe urethra. Direct electrical connection is established with theprostatic ductal and acinar epithelium via prostatic ducts that openinto the prostatic urethra. A surface electrode may then be placed perrectum onto the surface of the prostate and electrophysiologicalmeasurements may be made in a transepithelial fashion as described inthe breast. Similarly, provocative tests may be performed with drugs andhormones that differentially affect the electrophysiologicalcharacteristics of abnormal prostatic epithelium when compared to normalprostatic tissue.

Endometrial cancer may be diagnosed with an electrode cup placed on theuterine cervix. Physiological saline may be infused through the cervicalcanal to make electrical contact with the endometrium.Electrophysiological measurements may be made with a referenceelectrode, placed on the skin, intravenously or at a suitable referencepoint. Alternatively, this approach may be used during surgery where thecervical cup electrode is used in conjunction with a reference electrodeused on the peritoneal or outside surface of the uterus.

Salivary gland tumors open through small ducts into the oral cavity. Forexample in the parotid gland, Stensen's duct opens inside the mouthopposite the second upper molar tooth. A cup electrode may be used overthe opening of the duct inside the mouth. Physiological saline isinfused into the duct and electrical contact is thus established withthe ductal epithelium of the salivary gland. A surface electrode is thenused over the skin surface of the gland and electrical measurements areused to establish the diagnosis of cancer.

Although specific examples have been given above, this technique may beused to diagnose any tumor, where endoscopic access to the epithelium isnot possible or desired. The application of physiological saline via acup or short catheter may be used for example in the bowel or otherorgan system where electrical contact with the epithelium permits atransepithelial electrophysiological measurement to be made withoutresorting to endoscopic electrode placement. The second electrode isthen used to externally scan the organ for the presence of a tumor orabnormal epithelium. Since the physiological saline acts as an electrodein direct contact with the epithelium this approach simplifies theapproach to electrophysiological measurements. Depolarization and theimpedance characteristics of the epithelium will be more accurate whenthe surface-scanning electrode is in close proximity to the underlyingabnormal epithelium or tumor.

The embodiments described herein are described in reference to humans.However, cancers in non-humans may be also diagnosed with this approachand the present invention is also intended to have veterinaryapplications.

Any range of numbers recited in the specification hereinabove or in theparagraphs and claims hereinafter, referring to various aspects of theinvention, such as that representing a particular set of properties,units of measure, conditions, physical states or percentages, isintended to literally incorporate expressly herein by reference orotherwise, any number falling within such range, including any subset ofnumbers or ranges subsumed within any range so recited. Furthermore, theterm “about” when used as a modifier for, or in conjunction with, avariable, characteristic or condition is intended to convey that thenumbers, ranges, characteristics and conditions disclosed herein areflexible and that practice of the present invention by those skilled inthe art using temperatures, frequencies, times, concentrations, amounts,contents, properties such as size, surface area, etc., that are outsideof the range or different from a single value, will achieve the desiredresults as described in the application, namely, detectingelectrophysiological changes in pre-cancerous and cancerous tissue andepithelium, for example, breast tissue.

All documents described herein are incorporated by reference herein,including any patent applications and/or testing procedures. Theprinciples, preferred embodiments, and modes of operation of the presentinvention have been described in the foregoing specification.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the various aspects orembodiments of the present invention as set forth in the application andthe appended claims.

1. A method for determining a condition of a region of epithelial breasttissue comprising: (A) establishing a connection between a firstelectrode and the epithelial tissue of the nipple of a breast with aductal probe, an electroconductive media or both; (B) placing a secondelectrode in contact with the surface of the breast; (C) establishing asignal between the first and second electrodes; (D) establishing thatthe nipple ducts of the breast are open by: (1) measuring the impedancebetween the first and second electrode at about 5 different frequenciesin the range of about 200 Hz to about 60,000 Hz sufficient to establishan impedance curve; (2) treating the nipple using at least one methodselected from the group consisting of (a) applying suction and releaseof suction to the nipple; (b) applying alcohol; and (c) applying adekeratinizing agent; (3) again measuring the impedance between thefirst and second electrode at about 5 different frequencies in the rangeof about 200 Hz to about 60,000 Hz sufficient to establish an impedancecurve and comparing the impedance curve obtained to that obtained in (D)(1) above; (4) repeating steps (2) and (3) until the impedance curveobtained in step (D) (3) is substantially unchanged in order to confirmthat the ducts are open; (E) measuring between the first and secondelectrode: (1) a DC potential; and (2) impedance at about 5 differentfrequencies in the range of about 10 Hz to about 200 Hz and impedance atfrom about 5 to about 50 different frequencies in the range of about 0.1Hz to about 10 Hz; and (F) determining the condition of the region ofepithelial tissue based on the DC potential and impedance measurementsbetween the first and second electrode.
 2. A method for determining acondition of a region of epithelial breast tissue comprising: (A)placing over the nipple of a breast a cup having an interior, and firstand second openings, and an electrode disposed within the interior, thecup having a source of suction in communication with the first opening,the second opening having been placed over the nipple; (B) establishinga connection between the electrode and the epithelial tissue of thenipple of a breast with a ductal probe, an electroconductive media orboth; (C) placing a second electrode in contact with the surface of thebreast; (D) establishing a signal between the first and secondelectrodes; (E) establishing that the nipple ducts of the breast areopen by: (1) measuring the impedance between the first and secondelectrode at about 5 different frequencies in the range of about 200 Hzto about 60,000 Hz sufficient to establish an impedance curve; (2)treating the nipple using at least one method selected from the groupconsisting of (a) applying suction and release of suction to the nipple;(b) applying alcohol; and (c) applying a dekeratinizing agent; (3) againmeasuring the impedance between the first and second electrode at about5 different frequencies in the range of about 200 Hz to about 60,000 Hzsufficient to establish an impedance curve and comparing the impedancecurve obtained to that obtained in (D) (1) above; (4) repeating steps(2) and (3) until the impedance curve obtained in step (E) (3) issubstantially unchanged in order to confirm that the ducts are open; (F)measuring a DC potential between the first and second electrode; (G)applying suction to the cup sufficient to effect ductal collapse in anormal or non-malignant duct; and measuring impedance at about 5different frequencies in the range of about 10 Hz to about 200 Hz andimpedance at from about 5 to about 50 different frequencies in the rangeof about 0.1 Hz to about 10 Hz; and (H) altering the suction level andagain measuring impedance at about 5 different frequencies in the rangeof about 10 Hz to about 200 Hz and impedance at from about 5 to about 50different frequencies in the range of about 0.1 Hz to about 10 Hz; and(J) determining the condition of the region of epithelial tissue basedon the DC potential, and the impedance measurements under varyingpressure conditions.